C. limon Peels Based Nano-Bio pesticides: Formulation and Bioactivity against the Stored Pests and Microbes

C. limon Peels Based Nano-Bio pesticides: Formulation and Bioactivity against the Stored Pests and Microbes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article C. limon Peels Based Nano-Bio pesticides: Formulation and Bioactivity against the Stored Pests and Microbes Rabia bibi, Nazish Jahan, Kousar Rasheed, Samiah Samiah, Aqsa Hameed This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6714957/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Post-harvest losses due to stored grains being compromised by insects, microorganisms, and environmental factors pose a significant challenge. These losses canbe both quantitative, affecting nutritional value, and qualitative, leading to commercial loss. Effective pest control measures, including monitoring and early detection of infestations, are essential to protect stored grains.In this study, Citrus limon peels were selected for their biological activity and commercial viability and were processed into a nano-suspension. The acute toxicity and repellency of thesenano-formulations were tested against key stored product pests such as Oryzaephilus surinamensis , Tribolium confusum , and Sitophilus zeamais , alongside their antimicrobial activities. The developed nano-suspension exhibited optimal physical characteristics (particle size = 147.4 nm; PDI = 0.47). This formulation was repellent over time in area preference bioassays. Laboratory trials demonstrated that C. limon nano-suspension achieved a 100% mortality rate against O. surinamensis and S. zeamais , and an 80% mortality rate against T. confusum . Additionally, the C. limon peels-based nano-biopesticides exhibited high antimicrobial activity against bacterial pathogens ( Pseudomonas syringae and Clavibacter michiganensis ) and fungal pathogens ( Fusarium oxysporum and Rhizopus stolonifer ). These results indicate that C. limon peels-based nano-biopesticides, with their high dissolution rate, serve as effective antimicrobial agents and repellents, offering a promising alternative to chemical pesticides for controlling phytopathogenic species. Nanosuspension Citrus limon peels nano biopesticides Zeta sizer Morality rate Tween 80 stabilizer Particle size RSM Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 Figure 18 INTRODUCTION Pulses and cereals are attacked in stores by various insect pests. The storage insects are actually a major threat to storage grains and they lead to direct and indirect losses of cereal grains in storage (DEMIS and YENEWA, 2022). Since the beginning of agriculture and settled communities, grains have been the primary source of food for humans (Miedaner and Garbelotto, 2024). Some of the storage insect pests which attack storage grains are rice weevils, maize weevil, lesser grain borer, granary, weevil, red flour beetle, khapra beetle, and pulse beetles. In order to meet their needs for food and shelter, stored grain pests infest grains during storage, which results in both quantitative and qualitative losses (Chaubey, 2022). The indiscriminate usage of traditional pesticides has resulted in pesticide resistance, which also has an impact on soil fertility. Ultimately, there is a need to find alternative means for managing pest infestation (Summer et al. , 2024). Natural compounds and microorganisms have also been utilized as bio pesticides all over the world (Assadpour et al. , 2024). Despite, their progress have been increasing in recent decades, but bio pesticides use remains limited due to a lack of comprehensive knowledge of their modes of action, suitable storage and handling (Karabulut et al. , 2024). Other reasons for not considering bio pesticides as mainstream pest management tools include their relatively higher cost, lower stability, and reduced efficacy (Ferreyra-Suarez et al. , 2024). The research on sustainable and effective pest management has led the scientists to look for the innovative material solutions. Biodegradable and biocompatible substances with intelligent properties have the potential for developing safer pesticides (Abdollahdokht et al. , 2022). Nanotechnology offers a transformative approach by enabling the creation of precisely engineered pesticide delivery systems. These Nano-pesticides can be designed to release the active ingredients in a controlled (Vishnu et al. , 2024). Lemon is a valuable medicinal plant species of the Rutaceae family. Lemon is grown primarily for its alkaloids, which have anticancer properties and the bactericidal properties of crude extracts in different sections of the plant (such as the peels, flowers, stems, leaves, and roots) against clinically important bacterial strain (Zaman et al. , 2024). Citrus limon peels have antibacterial, antifungal, and antimicrobial properties. However, there are several limitations regarding the use of C. limon peels as bio pesticides, such as low solubility, instability, poor bioavailability, and slow release of active ingredients. The purpose of our present study is to formulate a C. limon nanosuspension to enhance the dissolution rate, control the release of active ingredients, and improve stability and bioavailability to increase its therapeutic properties against agricultural pests as a nano-biopesticide. After synthesis, the insecticidal activity of this nanosuspension was examined against economically important stored grain pests such as Tribolium confusum , Oryzaephilus surinamensis , and Sitophilus zeamais . The objectives of this study were, firstly, the formulation and optimization of an eco-friendly nanosuspension of Citrus limon peels with increased pest control efficacy, and secondly, the evaluation of the pesticidal and antimicrobial potential of the prepared nanosuspension. Results from laboratory trials showed the excellent efficacy of the C. limon nanosuspension and its promising insecticidal activity against the phyto-pathogenic species and microbes. Materials and Methods 2.1 Chemicals Citrus limon peels were sourced from a local market in Faisalabad, Pakistan. The following chemicals were procured for the study: ethanol (99.5% purity), polyvinyl pyrrolidone (PVP; 99% purity), poly vinyl alcohol (PVA; 99% hydrolyzed), hydroxyl propyl methylcellulose (HPMC; 99% purity), Tween 80 (99% purity), and polyethylene glycol (PEG; 99.5% purity) from Sigma-Aldrich (USA), and nutrient agar from Merck (Germany). 2.2 Collection of sample Citrus limon peels were used for the effective formation of the nano suspension. Lemon peels ( C. limon ) were collected from a local market in Faisalabad and cleaned to remove dirt and dust particles. The lemon peels were ground into a fine powder and stored in an airtight jar for subsequent studies. 2.3 Preparation of plant extract The dried plant sample was crushed into a fine powder using a Binatone MX10 blender. All compounds were extracted from the plants using a Soxhlet apparatus. To separate the desirable compounds from C. limon , ethanol was used as a solvent. The extraction process lasted for 6 to 8 hours, or until the intense yellow-green color of the lemon peels within the thimble became colorless. The extract of lemon peels was then filtered and concentrated for 2 to 3 hours using a rotary evaporator (Buchi, CH-9230 Flawil 1, Switzerland). After this, the concentrated extract was stored in the refrigerator (2 to 8°C) for further analysis (Liu et al. , 2024). 2.4 Formulation of nanosuspension 2.4.1 Screening of stabilizers The nanosuspension formulation included six distinct stabilizers: polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), sodium lauryl sulphate (SLS), Tween 80, hydroxyl propyl methyl cellulose (HPMC), and polyethylene glycol (PEG) (Park et al. , 2024). Throughout experiment, the solvent (ethanol) to anti-solvent (distilled water) ratio was kept at 10:20. An amount of 0.25 g of the each stabilizer was measured using a weighing balance and dispersed in 100 ml of water (anti-solvent) in 500 ml Pyrex flask. After that, 0.25 g of each extract of plant was added in 10 ml of suitable solvent (ethanol) and properly mixed. The mechanical shaker (RW 28 electronic overhead rotator, Thomas Research) was used for 6 hours around 4000 rpm. After stirring the mixture, each nano suspension was stored at room temperature (27 ± 3°C) or in the refrigerated (8–10°C) for one week to find the best stabilizer (Magi et al. , 2024). 2.4.2 Optimization of formulation parameters After stabilizers screening, different amounts of stabilizers and plant extract were optimized to formulate an effective nano suspension with minimum particle size. For all complete runs, a solvent to anti-solvent ratios (S/AS) were kept constant 10:20. Three various parameters including plant extract and amounts of stabilizers (0.25-1) as well as solvent to anti-solvent ratio (10–20) were optimized by Design Expert (Ver. 13) software along with two responses (particles size and PDI). The nano-precipitation method (bottom up approach) was used to produce C. limon nanosuspension (Cun et al. , 2021). The central composite design of RSM provided an experimental design for optimizing various parameters. 2.5 Characterization of nanosuspension Nanosuspension was characterized by employing following technique. 2.5.1 Particle size analysis The PDI (polydispersity index) and average particle size (Z-average-nm) of the synthesized C. limon nanosuspension were evaluated using a DLS (Dynamic Light Scattering 0012), a 12 mm cell through Zeta Sizer. The plant sample was taken in glass cuvettes. For each RSM prepared nanosuspension of C. limon peels, histogram was recorded (Perween et al. , 2021). 2.5.2 Stability studies To check the physical stability, C. limon nanosuspension was placed in refrigerator at 4 o C or at normal room conditions (25 o C) for 3 months. Less than 0.5 PDI and 100–600 nm particle size value proved the stability of nanosuspension. 2.6 Anti-microbial activity Antimicrobial activity of C. limon nanosuspension was investigated by well diffusion method. Two bacterial strains (gram positive Clavibacter michiganesis and gram negative Pseudomonas syringae ) were selected to analyze anti-bacterial activity of C. limon extract and its optimized nano suspension. 9.2 g (2.3 g in 100 ml) nutrient agar was dissolved in 400 ml distilled water. For sterilization, flask holding media was wrapped in aluminium foil and put in autoclave for 20 min at 121°C. This media was distributed evenly in petri-dishes (2–3 mm) in form of thin film gel. Wells were produced on agar medium, and 2ul of each bacterial strain was injected into different sterilised plates. At 25°C, every plate was incubated for 24 hours and zones of inhibition were measured (Anbazhagan, 2024). 2.7 Pesticidal activity C. limon peels extract was used in five different concentrations (50, 100 150, 200 and 250 mg/ml) along with control against three insects (red flour beetle, corn weevil and saw toothed beetle). Extracts of plant were also synthesized individually at very same concentration in distilled water in order to compare with nano suspension (Tran et al. , 2024). Simply, plastic petri dishes were utilized as enclosures for insects over Whatman No. 1 filter paper. 1ml of each plant extract, its nano suspension and control was sprinkled over each Whatman No. 1 filter paper and allowed to dry for 2 minutes under a fume hood. Twenty adult beetles of each pest were introduced into every petri dish and tightly wrapped using plastic tape. The petri dishes were then placed in an incubator. After 2, 48 and 72 hours, the percentage of insects that died was calculated (Grigg et al. , 2024). 2.7.1 Statistical analysis The efficacy of C. limon peels nano suspension and extract dilutions was determined using analysis of variance (ANOVA). All the trials were repeated three times, and Mortality rate of insects was calculated by Abbot's formula (Hasaballah et al. , 2021). Where, MC = control mortality and MO = observed mortality 2.8 Anti-fungal activity 16.8 g (4.2 g in 100 ml) of Potato Detrox Agar (PDA) was autoclaved in 400 ml of distilled water. For sterilization, the flask holding the media was wrapped in aluminium foil and put in autoclave for 20 min at 121°C (Rennick, 2023). The PDA medium was then put into the sterilized petri plates. In 1 ml of distilled water, two fungal strains ( F. oxysporum and R. stolonifer ) were added separately. PDA medium had already been poured into these sterile petri dishes. Wells were produced. The extract and its optimized nano suspension of various concentrations (150, 200, and 250 mg/ml) were poured into these wells. At 25°C, these petri dishes were incubated for 48 hours and zones of inhibition were measured after incubation (Morteza-Semnani et al. , 2021). 2.9 Statistical analysis For optimization studies, a central composite design using response surface methodology (RSM) was employed. The statistical experimental design was generated and analyzed using Design-Expert Software (Version 13). Analysis of variance was conducted with Tukey's test, applying a significance level of p < 0.05 (Agbangba et al. , 2024). RESULTS AND DISCUSSION 3.1 Screening of stabilizers Six distinct Food, Drug Administration approved and secure excipients, including SLS, Tween-80, PEG, PVP, PVA and HPMC were used as a stabilizer for synthesis of minimum sized C. limon nano suspension. For suitable stabilizer selection, 0.25 g plant extract was added in 0.25 g stabilizer at fixed solvent to anti-solvent ratio (1:10). For further experimentation, a stabilizer with the lowest particle size and PDI value was selected. Results of stabilizer screening for the formulation of C. limon nano suspensions are given in Table 1. Table 1 Screening of stabilizers for the formation of C. limon nanosuspension (Porwal, 2022) Nanosuspension Code Stabilizer Physical stability Z-average size Polydispersity index Stability after 3 month (room temperature 1 PVA Stable 129 0.5 Unstable 2 Tween 80 Stable 147 0.4 Stable 3 PEG Unstable 580 1 Unstable 4 SLS Unstable 557 1 Unstable It was apparent from the result that all formulated C. limon nano suspensions with HPMC, PVA, PVP, tween-80 and PEG stabilizers were found physically stable when freshly prepared. But nano suspensions with HPMC and PVP stabilizers lost stability after one week (figure S1 in supplementary file). Therefore, only four nano suspensions were characterized. After characterization, nano suspension with PEG, SLS, PVA stabilizers were also lost stability after 3 month at room temperature (25 o C). While nano suspension formulated with Tween-80 stabilizer remained stable even after 3 months. But Tween 80 stabilizer was selected for further optimization due to greater stability rate (Fig.S.6 in supplementary file). Figure 1 below shows particle size and PDI value of C. limon nano suspension with Tween 80 stabilizer 3.2 Optimization of parameter s of C. limon nano suspension by response surface methodology (RSM) In order to formulate therapeutically stable and efficient nanosuspension with the desired morphology and particle size, optimization of formulation parameters is required. After selecting a suitable stabilizer, various concentrations of the plant extract and stabilizer were optimized to achieve a nanosuspension with minimal particle size. The nano suspensions were formulated using a modified nano precipitation method (bottom-up approach). The formulation parameters, including the amount of plant extract (A), stabilizer concentration (B), and the S/AS ratio (C), were optimized through the central composite design of RSM. Optimization was focused on achieving the smallest particle size and lowest PDI value. Based on the response values obtained, the software predicted a quadratic model for the nanosuspension particle size. The influence of the independent variables on the response values were assessed by developing a regression equation for the response (Table S.2 and S.3 in supplementary file). Citrus limon particle size (nm) (R1) = + 672.46 + 67.66A + 77.28B + 11.39C + 36.59AB + 79.56AC − 122.29BC − 109.91A 2 − 1 53.41 − 2 7.10C 2 (1) Citrus limon PDI (R2) = + 0.1704–0.0854A + 0.0176B -0.0503C -0.1862AB -0.0617AC + 0.0383BC + 0.1684A 2 + 0.1394B 2 + 1861C 2 (2) The positive coefficients indicate a combined effect of the independent variables on reduction of particle size and PDI, while the negative values denotes an inverse effect. From the equations, it is evident that the amount of plant extract, concentration of stabilizer, solvent to anti-solvent ratio, and their interactions play crucial roles in decreasing the particle size and polydispersity index (PDI) of the C. limon nanosuspension (Table . ANOVA was utilized to assess the linear, quadratic, and interactive effects of the independent variables on R1 and R2. The p-values (p = 0.0001) and F-values (61.93 for R1 and 88.20 for R2) demonstrated the significance of the quadratic model for optimizing the nanosuspension synthesis. P-values were used to determine the model's significance, with a p-value of < 0.05 indicating a significant model. The variables A and B, along with the interactions AC, BC, and the quadratic terms A² and B², had a significant impact on reducing the particle size of the C. limon nanosuspension. Additionally, the parameters A, C, AB, AC, BC, A², B², and C² significantly affected the reduction in PDI, while the remaining parameters showed an inverse relationship with particle size and PDI (Tables 2 and 3). Table 2 ANOVA for quadratic model of particle size (R1) of C. limon nano suspension (Uchida et al. , 2021) Source Sum of Squares df Mean Square F-value p-value Model 7.967E + 05 9 88523.94 61.93 < 0.0001 Significant A-Amount of plant extract 62526.86 1 62526.86 43.74 < 0.0001 B-Amount of Stabilizer 81558.48 1 81558.48 < 0.0001 C-Solvent to anti-solvent ratio 1771.48 1 1771.48 1.2457.5 0.2917 AB 10710.62 1 10710.62 7.49 0.0209 AC 50644.71 1 50644.71 35.43 0.0001 BC 1.196E + 05 1 1.196E + 05 83.69 < 0.0001 A² 1.741E + 05 1 1.741E + 05 121.78 < 0.0001 B² 3.393E + 05 1 3.393E + 05 237.32 < 0.0001 C² 10587.57 1 10587.57 7.41 0.0215 Residual 14295.21 10 1429.52 Lack of Fit 7025.07 5 1405.01 0.9663 0.5145 not significant Pure Error 7270.13 5 1454.03 Cor Total 8.110E + 05 19 R² 0.9824 C.V. % 7.97 Predicted R² 0.9148 Adeq. Precision 21.0933 Adjusted R² 0.9824 The strong predictability or consistency of the model was suggested by the non-significant lack of fit F values of R1 (0.96) and R2 (0.54). The coefficient of determination (R 2 ) was used to provide an additional evaluation of the quadratic model's quality. The R 2 values for PDI (0.9876) and particle size (0.9824) indicated 98% variability in both responses. Because R2 was nearer 1.000, the model was more reliable and anticipated a better response. Regression models with R 2 > 0.9000 have historically been considered to have extremely strong correlation (Wu et al. , 2023). According to Tables 2 and 3, the CV for R1 and R2 was determined to be 7.97 and 8.43%, respectively, and was considered satisfactory (Table S.4 in supplementary file). Table 3 ANOVA for quadratic model of PDI (R2) of C. limon nanosuspension (Hazafa et al. , 2022) Source Sum of Squares Df Mean Square F-value p-value Model 1.45 9 0.1615 88.20 < 0.0001 Significant A-Amount of plant extract 0.0995 1 0.0995 54.35 < 0.0001 B-Amount of Stabilizer 0.0042 1 0.0042 2.30 0.1604 C-Solvent to anti-solvent ratio 0.0345 1 0.0345 18.85 0.0015 AB 0.2775 1 0.2775 151.54 < 0.0001 AC 0.0305 1 0.0305 16.66 0.0022 BC 0.0117 1 0.0117 6.39 0.0300 A² 0.4088 1 0.4088 223.24 < 0.0001 B² 0.2802 1 0.2802 153.00 < 0.0001 C² 0.4991 1 0.4991 272.56 < 0.0001 Residual 0.0183 10 0.0018 Lack of Fit 0.0065 5 0.0013 0.5499 0.7362 not significant Pure Error 0.0118 5 0.0024 Cor Total 1.47 19 R² 0.9876 C.V. % 8.43 Predicted R² 0.9549 Adeq. Precision 27.5240 Adjusted R² 0.9764 The impact of all independent variables on different responses of the C. limon nano suspension was examined by using three dimensional response surface plots. In every plot, combined effect of two variables was simultaneously evaluated, whereas a third one was kept as constant. These plots evaluated that all three formulation parameters have a significant effect on reduction in particle size and PDI of C. limon nanosuspensions (Table S.5 in supplementary file). 3.2.1 Impact of amount of plant extract and conc. of stabilizer on particle size of C. limon nanosuspension The graph clearly showed that when the stabilizer concentration decreased from 1 to 0.25g, there was a significant decrease in particle size. However, by increased the plant extract from 0.25g to 1 g, a significant decrease in particle size was also observed. Particle size (96.88 nm) significantly decreased at optimized conditions (1g plant extract and 0.25g concentration of stabilizer) by keeping solvent to anti-solvent ratio constant as showed in Fig. 2. A gradual increase in particle size was also examined by increasing the concentration of stabilizer from 0.25g. The explanation for decreasing particle size by increasing the polymer stabilizer concentration was that, an excess accumulation of polymer would enhance particle size and prevent the absorption during precipitation between solvent and anti-solvent. In addition, osmotic pressure increases by increasing the concentration of polymer. This leads to increased colloidal particle attraction, leading to greater size (Zhou et al. , 2023). These results were correlated with previous findings like particle size of nanosuspension was increased by increasing the amount of plant extract and concentration of stabilizer from optimized conditions (Hou et al. , 2022). 3.2.2 Impact of amount of plant extract and conc. of stabilizer on PDI of C. limon nanosuspension From the graph, it was observed that minimum PDI value was observed at 0.25 g stabilizer concentration and 1 g amount of plant by keeping solvent to anti-solvent ratio constant (Fig. 3). When we increased stabilizer concentration and amount of plant extract from optimized conditions (0.25 g conc. of stabilizer and 1 g plant extract), PDI value also increased. Results of the current study correlated with previous findings which showed that optimized concentration of stabilizer control PDI value and minimum PDI was observed under optimum concentration of stabilizer (Van Driessche et al. , 2022). 3.3 Biological activities of C. limon nanosuspension The following in-vitro biological assays were performed to check positive effective role of nanosuspension against bacteria, fungi and pests of agricultural origin. 3.3.1 Pesticidal activity The repellent and pesticidal activity of C. limon nanosuspension was observed against three pests like Tribolium castaneum (red flour beetle), Oryzaephilus surinamensis (sawtoothed grain beetle) and Sitophilus zeamais (corn weevil) under lab conditions. All treatments as well as control was applied in form of three replications. The C. limon nanosuspension, the coarse peels extract and Malathion (control) were applied in five different concentrations such as 50, 100, 150, 200 and 250mg/ml while distilled water was used as negative control. After 24, 48 and 72 hours of treatment applications, mortality data was examined for three days. Sitophilus zeamais or corn weevil is one of the primary stored grain pests. It mainly feeds on corn and damage crops like corn, grain, and cotton. Oryzaephilus surinamensis is a common secondary pest of cereals and cereal products and mostly found on copra, spices, nuts and dried fruit. Their adults and larvae are able to enter small cracks, so they can often attack packaged food or nuts in shell. Tribolium castaneum causes large economic losses of stored wheat grain. The major strategy used to protect stored crops is to employed chemicals in the form of pesticides. But excessive use of chemical insecticides caused environmental pollution and makes insect-resistant to a number of insecticides (Rauf, 2024). In this context, a new strategy should be analyze to combat major drawbacks associated with conventional insecticides. Due to enhanced rate of dissolution and solubility, increased stability and improved efficiency, nano suspensions become more reactive and this is one of the reasons for which it used as pesticides (Rani et al. , 2023). Pesticides typically function by inhibiting or disrupting the translation process and protein synthesis, often through binding to prokaryotic ribosomes, which prevents peptide transfer and prevent chain elongation (Kocyigit et al. , 2023). Nano bio pesticides have some added advantages over chemical and bio pesticides because of their increased solubilization abilities, bioavailability, stability, slow release, and targeted delivery, leading to enhanced efficiency. Studies on nano-formulations used against Tribolium castaneum have shown muscular destruction, pigmentation changes, and alterations in epidermal thickness, necrosis in the cuticle, and cellular damage in both the endocuticle and exocuticle. Ingestion of nano-biopesticides during early larval stages has been linked to impaired crawling and climbing abilities in later larval and adult stages (Bihal et al. , 2023). Due to their bioavailability in plant systems, nano-biopesticides are valuable for understanding the interactions and behaviors of various pests that infest crops (Bihal et al. , 2023) 3.3.2. Effect of C. limon nanosuspension against sawtoothed beetle ( O. surinamensis ) and red flour beetle ( T. castaneum ) Effect of C. limon nano suspension examined against saw-toothed beetle, red flour beetle and corn weevil to check the mortality rate of all thee insects. Twenty homogeneous adults were released in each petri-plate. These adults were allowed to feed on treated diet and mortality data was observed. The five varying concentrations were employed such as 50, 100, 150, 200, 250 mg/ml against saw-toothed beetle and red flour beetle. Three varying concentrations were employed such as 150, 200, 250 mg/ml against S. zeamais and mortality data was examined at 24, 48 and 72 hours of exposure time. Figures 4, 5 and 6 shows Pesticidal activity of C. limon peels coarse extract, C. limon nanosuspension and Malathion (control) against T. castaneum respectively The mortality rate of C. limon nanosuspension against O. surinamensis and T. castaneum at various treatment and exposure of times showed in Table 4 and 5. C. limon nanosuspension possessed significantly (p < 0.05) higher pesticidal activity than coarse peel extract against T. castaneum . The maximum mortality rate of 97% was observed after 72 hours of exposure time whereas minimum mortality of 62% was observed for nanosuspension after 24 hrs. of exposure time at 250 mg/ml concentration. Similarly, at 250 mg/ml, the highest mortality rate of 45% was recorded for the coarse peel extract after 72 hours, compared to the lowest rate of 29% observed after 24 hours. This indicates that prolonged exposure leads to a gradual increase in insect mortality. Previous research showed that D-limonene could interfere with the pests octopaminergic system and cause nervous system damage (Ibrahium et al. , 2022). The results of current investigation correlate with earlier reports in literature such as C. limon EO-based nano emulsions were most efficient against T. castaneum larvae by exhibiting 93.3% mortality rate in 14 days post-exposure (Papanikolaou et al. , 2022). Figures 7, 8 and 9 shows Pesticidal activity of C. limon peels coarse extract, C. limon nanosuspension and Malathion (control) against O. surinamensis respectively. Figures 10, 11 and 12 shows Pesticidal activity of C. limon peels coarse extract, C. limon nanosuspension and Malathion (control) against S. zeamais respectively. C. limon nanosuspension also possessed significantly (p < 0.05) higher pesticidal activity than coarse peel extract against O. surinamensis. The maximum mortality of 100% was observed after 24 hours of exposure time at highest concentration 250 mg/ml, whereas minimum mortality of 14% was observed after 24 hours of exposure time at lowest conc. of 50 mg/ml. Similarly, maximum mortality of 26% was recorded for the coarse peel extract after 72 hours, compared to the lowest rate of 15% observed after 24 hours. These results were correlated with the previous findings which showed that the mortality rate of pests was also increased gradually by increasing concentrations of nanosuspension (Palermo et al. , 2021). But Malathion (control) showed significantly (p < 0.05) higher mortality rate than both C. limon nanosuspension and coarse peels extract against O. surinamensis and T. castaneum and S. zeamais as showed in Fig. 6. Table 4 Effect of different exposure time and concentrations of C. limon nanosuspensions and peels extract on mortality of T. castaneum (El-Gendy et al. , 2024) Exposure time (h) Conc. mg/ml % Average Mortality ± SE Nanosuspension Plant extract Positive control Negative control 24 5 10.0 Ct ± 0.5 6.66 AcaCt ± 0.3 13.33 Ct ± 0 0.00 Cu 10 16.66 Ct ± 0.3 13.33 Ct ± 0.3 18.33 Ct ± 0.3 0.00 Cu 15 38.33 Copqr ± 0.3 30.0 Crs ± 0.57 41.6 Cnopq ± 0.3 0.00 Cu 20 58.33 Cijk ± 0.3 35.0 Cpqr ± 0.57 61.6 Chijk ± 0.3 0.00 Cu 25 76.33 Cef ± 0.3 56.66 Cijk ± 0.3 78.3 Cdef ± 0.3 0.00 Cu 48 5 31.66 Bqrs ± 0.3 21.66 Bst ± 0.3 33.3 Bpqr ± 0.3 0.00 Bu 10 38.33 Bopqr ± 0.3 28.33 Brst ± 0.3 43.3 Bmnop ± 0.5 0.00 Bu 15 61.66 Bhijk ± 0.3 41.66 Bnopq ± 0.3 63.33 Bghij ± 0.3 0.00 Bu 20 73.33 Befg ± 0.3 51.66 Bklmn ± 0.6 78.33 Bdef ± 0.3 0.00 Bu 25 83.33 Bcde ± 0.3 61.66 Bhijk ± 0.3 88.33 Bbcd ± 0.3 0.00 Bu 72 5 51.66 Aklmn ± 0.3 36.33 Aopqr ± 0.3 56.6 Ajkl ± 0.57 0.00 Au 10 71.66 Afgh ± 0.3 46.6 Almno ± 0.3 75.0 Aef ± 0.57 0.00 Au 15 78.33 Adef ± 0.3 53. 33Ajklm ± 0.3 83.3 Acde ± 0.57 0.00 Au 20 91.66 Aabc ± 0.3 61.66 Ahijk ± 0.3 93.3 Aabc ± 0.57 0.00 Au 25 98.66 Aab ± 0.3 68.33 Afghi ± 0.3 100 Aa + 0.57 0.00 Au The % average mortality is given as mean ± SD. Means following same uppercase letter in each column are not substantially different. Within every row, means followed by the same lowercase letter are not significantly different; Tukey HSD test, p ˂ 0.05. Table 5 Effect of different exposure time and concentrations of C. limon nanosuspensions and peels extract on mortality of O. surinamensis (Jampílek et al ., 2020) Exposure time (h) Conc. mg/ml % Average Mortality ± SE Nanosuspension Plant extract Positive control Negative control 24 50 23.33 Cqr ± 0.3 13.33 Cr ± 0.3 28.33 Copq ± 0 0.00 Ct 100 38.33 Clmno ± 0.3 25.0 Cpqr ± 0.57 43.3 Cjklm ± 0.57 0.00 Ct 150 71.66 Cdef ± 0.3 33.3 Cmnopq ± 0.3 75.0 Ccde ± 0.3 0.00 Ct 200 100 Ca ± 0 46.66 Cijkl ± 0.3 100 Ca ± 0.3 0.00 Ct 250 100 Ca ± 0 55.0 Cghij ± 0.57 100 Ca ± 0.3 0.00 Ct 48 50 33.33 Bmnopq ± 0.3 25.0 Bpqr ± 0.3 36.6 Blmnop ± 0.3 0.00 Bt 100 58.33 Bghi ± 0.3 31.6 Bmnopq ± 0.6 60.0 Bfgh ± 0.57 0.00 Bt 150 76.66 Bbcd ± 1.0.3 41.66 Bklmn ± 0.3 78.33 Bbcd ± 0.3 0.00 Bt 200 100 Ba ± 0 55.0 Bghij ± 0.57 100 Ba ± 0.3 0.00 Bt 250 100 Ba ± 0 61.66 Bfg ± 0.3 100 Ba ± 0.3 0.00 Bt 72 50 51.66 Aghijk ± 0.3 30.0 Anopq ± 0.57 53.3 Aghijk ± 0.3 0.00 At 100 78.33 Abcd ± 0.3 41.66 Aklmn ± 0.8 80.0 Abcd ± 0.3 0.00 At 150 86.66 Abc ± 0.3 48.33 Ahijkl ± 0.8 88.33 Aab ± 0.3 0.00 At 200 100 Aa ± 0 63.33 Aefg ± 0.3 100 Aa ± 0.3 0.00 At 250 100 Aa ± 0 71.66 Adef ± 0.3 100 Aa ± 0.3 0.00 At The % average mortality is given as mean ± SD. Means following same uppercase letter in each column are not substantially different. Within every row, means followed by the same lowercase letter are not significantly different; Tukey HSD test, p˂ 0.05. 3.3.3. Effect of C. limon nanosuspension on corn weevil ( S. zeamais ) The study revealed that C. limon nanosuspension was also much potent against insect S. zeamais and possessed significantly (p < 0.05) higher pesticidal activity than coarse peel extract. The maximum mortality of 100% was observed after 24 hours at highest concentration 250mg/ml, whereas minimum mortality rate 25% was observed after 24 hours of exposure time at 150mg/ml concentration. Similarly, after 72 hours of treatment with coarse peel extract, the mortality rate reached 23%. After 24 hours, the mortality rate was 10%. This indicates that longer exposure leads to a gradual increase in S. zeamais mortality. But Malathion (control) showed significantly (p < 0.05) higher mortality rate than both Citrus limon nanosuspension and coarse peels extract against S. zeamais . Table 6 shows Effect of different exposure time and concentrations of C. limon nanosuspensions and peels extract on mortality of S. zeamais Table 6 Effect of different exposure time and concentrations of C. limon nanosuspensions and peels extract on mortality of S. zeamais (Gadelhaq et al. , 2023) Exposure time (h) Conc. mg/ml % Average Mortality ± SE Nanosuspension Plant extract Positive control Negative control 24 150 41.66 Cde ± 0.3 26.66 Cj ± 0.57 43.33 Ccde ± 0.3 0.00 Ck 200 78.33 Ccde ± 0.3 48.33 Cj ± 0.57 80.0 Ccd ± 0.3 0.00 Ck 250 100 Ccd ± 0 58.33 Cj ± 0.57 100 Cbcd ± 0.3 0.00 Ck 48 150 71.66 Bbcd ± 0.3 38.33 Bi ± 0.57 73.33 Babcd ± 0.3 0.00 Bk 200 88.33 Bab ± 0.3 61.66 Bhi ± 0.57 90.0 Babc ± 0.3 0.00 Bk 250 100 Ba ± 0.3 71.66 Bghi ± 0.5 100 Ba ± 0.3 0.00 Bk 72 150 78.33 Aabc ± 0.57 53.33 Agh ± 0.3 80.0 Aa ± 0.57 0.00 Ak 200 91.66 Aa ± 0.57 70.0 Afg ± 0.57 93.33 Aa ± 0.3 0.00 Ak 250 100 Aa ± 0 78.33 Aef ± 0.3 100 Aa ± 0.3 0.00 Ak The % average mortality is given as mean ± SD. Means following same uppercase letter in each column are not substantially different. Within every row, means followed by the same lowercase letter are not significantly different; Tukey HSD test, p ˂ 0.05. 3.4 Antibacterial activity The antibacterial activity of C. limon peels extract and their optimized nanosuspensions were investigated using the well diffusion method. Two bacterial strains such as Clavibacter michiganesis (gram-positive) and Psedomonas syringae (gram negative) were used for this purpose. For antibacterial activity, Cciprofloxacin was used as a control while distilled water was used as negative control .The results were recorded in the absence or presence of an inhibition zone around the well. Each treatment was replicated three times. After treatments application, zone of inhibition were observed after 24 hours of exposure time. The antimicrobial activity was performed using three varying concentrations such as 150, 200, 250 mg/ml against two strains of bacteria such as Pseudomonas syringe and Clavibacter michiganesis . Bacterial speck, caused by P syringae pathovar tomato (Pst), is a significant threat to tomato crops, potentially leading to a 75% yield loss if infection occurs during the seedling stage. The extensive use of chemical bactericides to manage this disease has led to pathogen resistance, environmental contamination, and health risks (Wang et al. , 2023). Therefore, C. limon nanosuspension has demonstrated effective results against this strain in preventing plant disease. Additionally, C. michiganensis , the pathogen responsible for bacterial ring rot in potatoes ( Solanum tuberosum ), is a notorious plant pathogen that has severely impacted potato cultivation. The extract from C. limon peels has shown promising efficacy against this bacterial strains (Osdaghi et al ., 2022). Table 7 Anti-bacterial activity against P. syringae and C. michiganesis (Arredondo-Valdés et al ., 2020) Antimicrobial activity Concentration (µg/ml) Zone of inhibition (mm) P. syringae C. michiganesis C. limon Plant Extract 150 8.46 Cg ± 0.2 5.6 Ci ± 0.15 200 10.4 Cf ± 0.1 10.46 Ch ± 0.25 250 15.7 Ce ± 0.2 12.53 Cg ± 0.25 C. limon Nanosuspension 150 18.4 Be ± 0.2 15.23 Bf ± 0.20 200 20.5 Bc ± 0.2 18.13 Be ± 0.15 250 22.13 Bb ± 0.15 20.2 Bc ± 0.2 Positive Control 150 21.4 Ad ± 0.1 17.26 Ad ± 0.15 200 24.8 Ab ± 0.1 20.33 Ab ± 0.15 250 26.7 ± 0.25 22.6 Aa ± 0.1 Negative Control 150 0.00 Dh 0.00 Dj 200 0.00 Dh 0.00 Dj 250 0.00 Dh 0.00 Dj The zone of inhibition is given as mean ± SD. Means following same uppercase letter in each column are not substantially different. Within each row, means followed by the same lowercase letter are not significantly different; Tukey HSD test, p ˂ 0.05. The results of antibacterial activity showed that C. limon nanosuspension showed significantly (p < 0.05) higher antibacterial activity against Pseudomonas syringae and Clabibacter michiganesis than peels extract of same concentration (Tables 7 and 8). C. limon nanosuspension possessed significantly (p < 0.05) higher inhibition zone 22.13 ± 0.15 than C. limon coarse peels 15.7 ± 0.2 whereas positive control Ciprofloxacin possessed maximum zone of inhibition 26.7 ± 0.25 against Pseudomonas . Ciprofloxacin showed significantly (p < 0.05) higher antibacterial activity (26.7 ± 0.25) in comparison to all other treatment groups in case of both strains. In case of C. michiganesis (gram positive strain), maximum zone of inhibition manifested by C. limon nanosuspension was 20.2 ± 0.2 and C. limon coarse peels extract had 12.53 ± 0.25 zone of inhibition, whereas Ciprofloxacin (control) possessed 22.6 ± 0.1 zone of inhibition. This was clearly suggested that even low dosage of plant based nanosuspension has worked great and much compatible with market based product. Ciprofloxacin showed significantly (p < 0.05) higher anti-bacterial activity against P. syringae and C. michiganesis in comparison to all other treatment groups in case of both strains. 250mg/ml concentration of nano suspension was found out more effective against pesticides than other concentrations of nano suspension. Figures 13, 14 and 15 shows Zone of inhibition for C. limon peels extract, C. limon nanaosuspension and Ciprofloxacin (control) against P. syringae and C. michiganesis Higher antibacterial activity of nanosuspensions can be due to higher dissolution rate and improved diffusion of nanosuspension in cultural media during experimental growth of bacteria (Singh et al. , 2024). The strong reactivity and distinctive interactions of nanosuspensions with biological systems are a result of their distinctive physicochemical features (ultra-small size, large surface to mass ratio) (Marques and Kumar, 2023). This was concluded that the nanosuspension of C. limon had quite comparable results with commercial antibiotics like Ciprofloxacin. 3.5. Antifungal activity Antifungal activity of C. limon peels extract and their optimized nanosuspension was evaluated against two fungal strains such as Fusarium oxysporum and Rhizopus stolonifer by well diffusion method. The antifungal activity of C. limon extract, its optimized nanosuspension and standard control (Iitraconazole) were evaluated at three different concentration such as 150, 200, 250 mg/ml. Iitraconazole was used as positive control while distilled water was used as negative control. 4.2g/100ml of Potato Dextrose Agar (PDA) was added in petri dishes and inoculated with fungal strains. Appropriately wells were impregnated with 30µl samples of treatment. The antifungal activities of treatments were examined after 48 hours by measuring zone of inhibition. The results of antifungal activity of optimized nanosuspension and coarse peels extract against both strains showed in Table 8. C. limon nanosuspension possessed significantly (p < 0.05) higher antifungal potential than peels extract and control againt F. oxysporum and R. stolonifer . The results showed that C. limon nanosuspension possessed maximum inhibition zone (25.46 ± 0.40) against F. oxysporum at 250mg/ml concentration and minimum zone of inhibition (19.46 ± 0.25) was observed at 50mg/ml concentration than C. limon coarse peels 18.6 ± 0.25 whereas positive control Ciprofloxacin possessed maximum zone of inhibition 0.83 ± 0.25 against F. oxysporum . In case of R. stolonifer , maximum zone of inhibition manifested by C. limon nanosuspension was 20.12 ± 0.1 and C. limon coarse peels extract had 13.33 ± 0.1 zone of inhibition, whereas Ciprofloxacin (control) possessed 0.96 ± 0.25 zone of inhibition. Whereas, Fluconazole and Voriconazole (eminent antimycotic drugs) were used as controls, but surprisingly, they didn’t show any invitro antimycotic activity. But it was later found from a literature survey that the fungal species have developed resistance against the antimycotic drugs that were being used as antifungal control (Gupta et al. , 2024). So, Itraconazole was showed better results as an antifungal control. Itraconazole showed significantly (p < 0.05) lower antibacterial activity in comparison to all other treatments in case of both strains Zone of inhibition increased with increased in the concentrations of C. limon nanosuspension. Our results were closed to previous findings which examined that C. limon peels extract caused 76.67% zone of inhibition against F. oxysporum at 500 mg/ml (Gautam and Khedkar, 2024). Table 8 Anti-fungal activity against F. oxysporum and R. stolonifer (Badaracco et al. , 2020) Antimicrobial activity Concentration (µg/ml) Zone of inhibition (mm) F. oxysporum R. stolonifer C. limon Plant Extract 150 14.5 Ch ± 0.2 9.33 Ce ± 0.1 200 16.35 Cg ± 0.2 11.53 Cd ± 0.1 250 18.63 Cf ± 0.25 13.33 Cc ± 0.1 C. limon Nanosuspension 150 19.46 Be ± 0.25 16.15 Bi ± 0.1 200 21.46 Bd ± 0.30 18.42 Bh ± 0.1 250 25.46 Bb ± 0.40 20.12 Bg ± 0.1 Positive Control 150 0.78 Aa ± 0.26 0.73 Aa ± 0.26 200 0.87 Aa ± 0.37 0.85 Aa ± 0.37 250 0.90 Aa ± 0.25 0.96 Aa ± 0.25 Negative Control 150 0.00 Di 0.00 Dj 200 0.00 Di 0.00 Dj 250 0.00 Di 0.00 Dj The zone of inhibition is given as mean ± SD. Means following same uppercase letter in each column are not substantially different. Within every row, means followed by the same lowercase letter are not significantly different; Tukey HSD test, p ˂ 0.05. It was concluded from results that C. limon nanosuspension possessed greater zone of inhibition against F. oxysporum and R. stolonifer than coarse peels extract. 250mg/ml concentration of nanosuspension was found out more effective against pesticides than other concentrations of nanosuspension. The biological active constituent of C. limon peels is responsible for antifungal activity (Hou et al. , 2022). Hence, plant-based nanosuspensions can be used as antifungal agents more effectively than commercial drugs due to their equivalent outcomes, cost effectiveness, high solubility, and ease of degradability. So, Present research work is in accordance with the previous study where the ultimate aim of researcher was to develop plant based formulation for treating plant disease and safe storage of grains (Albahri et al. , 2023). Our result were closed to previous study which studied that turmeric essential oil showed antifungal activity against mycelial growth and spore germination of A. flavus in vitro (DEMIS and YENEWA, 2022). Conclusion In conclusion, the conventional use of chemical pesticides has significantly improved agricultural production but has raised environmental concerns. Nano biopesticides presents a promising solution, combining efficient pest control with enhanced properties such as durability and biodegradability. This study successfully formulated a nanosuspension of C. limon using the nanoprecipitation method, demonstrating its positive impact on the health and growth of different plants in pot trials. The nanosuspension exhibited strong pesticidal activity highlighting its potential as an environmentally friendly and effective alternative for sustainable plant protection. With its notable stability and biodegradability, C. limon plant-based nanosuspension emerges as a preferable choice for future agricultural practice. Declarations Ethics Declaration Conflict of Interest The authors declare the following financial interests/personal relationships which may be considered as potential conflict of interest. Ethical approval University has allowed us to conduct research on insects and pests in limited resource for wellbeing of human as well as for environment. During this research, anti-bacterial and anti-fungal activities were performed by using C. limon nanobiopesticides. Funding: This study has been funded by Endowment Fund Secretariat University of Agriculture, Faisalabad, Pakistan, under grant No. RD-028-18. Data Availability: The following details on the availability of data were provide: The supplementary Files provide the raw measurements. Consent for publications: All authors agree to publish. Consent to Participate: Not applicable for this section. Competing interests: The authors declare no competing interests. Ethics Declaration: Before participating in the study, every participant gave their informed permission, and the research was carried out in compliance with ethical guidelines and authorized by the appropriate institutional review board. Acknowledgements: Hereby, we extend our gratitude to A. Q Research Group, Pakistan for reviewing the article and providing helpful comments. Author contribution All authors contributed to the study conception and design. Methodology, data duration, investigation, and software were performed by Rabia Bibi. Project administration, visualization, and supervision were performed Nazish Jahan. Conceptualization, methodology, investigation, and validation were performed by Rabia Bibi. Review and editing were performed by Kousar Rasheed, Samiah Samiah and Aqsa Hameed. The first draft of the manuscript was written by Rabia Bibi, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Abdollahdokht, D., Y. Gao, S. Faramarz, A. Poustforoosh, M. Abbasi, G. Asadikaram and M. H. Nematollahi. 2022. Conventional agrochemicals towards nano-biopesticides: an overview on recent advances. Chem. Biol. Technol. Agric. 9:1-13. Agbangba, C. E., E. S. Aide, H. Honfo and R. G. Kakai. 2024. On the use of post-hoc tests in environmental and biological sciences: A critical review. Heliyon 1:20-40. Aisvarya, S., M. Kalyanasundaram, M. Kannan, P. Arunkumar, S. Preetha, K. Elango and K. Govindaraju. 2024. Comparative analysis of the insecticidal activity against Sitophilus oryzae (L.) and agro-morphological characteristics of maize using non-biogenic and biogenic ZnO nanoparticles. Environ. Sci. Nano, 11:2173-2187. Akhavan-Mahdavi, S., R. Sadeghi, A. F. Esfanjani, S. Hedayati, R. Shaddel, C. Dima, N. Malekjani, S. Boostani and S. M. Jafari. 2022. Nanodelivery systems for d-limonene; techniques and applications. Food Chem. 384:132460-132479. Albahri, G., A. A. Alyamani, A. Badran, A. Hijazi, M. Nasser, M. Maresca and E. Baydoun. 2023. Enhancing essential grains yield for sustainable food security and bio-safe agriculture through latest innovative approaches. Agronomy 13:1709-1715. Almahdy, A. G., A. El-Sayed and M. Eltarahony. 2024. A novel functionalized CuTi hybrid nanocomposites: facile one-pot mycosynthesis, characterization, antimicrobial, antibiofilm, antifouling and wastewater disinfection performance. Microb. Cell Fact. 23:140-148. Anbazhagan, S. 2024. Biophysical characterization of Brucella abortus S19 perand evaluation of cross protective efficacy in mice model. Indian Veterinary Research Institute. Arredondo-Valdés, R., J. C. Chacón-Hernández, F. Reyes-Zepeda, F. D. Hernández-Castillo, J. C. Anguiano-Cabello, R. T. Heinz-Castro and S. G. Mora-Ravelo. 2020. In vitro antibacterial activity of Magnolia tamaulipana against tomato phytopathogenic bacteria. Plant Prot. Sci. 56:4-10 Assadpour, E., A. Can Karaça, M. Fasamanesh, S. A. Mahdavi, M. Shariat-Alavi, J. Feng, M. S. Kharazmi, A. Rehman and S. M. Jafari. 2024. Application of essential oils as natural biopesticides; recent advances. Crit. Rev. Food Sci. Nutrit. 64:6477-6497. Elmadawy, A. A., D. M. El-Talpanty, M. El-Ebiary, T. Ismail and A. F. Omar. 2024. Toxicity and morphological changes induced by diatomaceous earth & malathion against Tribolium castaneum. New Valley J. Agric. Sci. 4:1-12. Babarinde, S. A., K. A. Kemabonta, O. Z. Olatunde, E. O. Ojutiku and A. K. Adeniyi. 2021. Composition and toxicity of rough lemon (Citrus jambhiri Lush.) rind essential oil against red flour beetle. Acta Ecol. Sin. 41: 325-331. Badaracco, P., M. Sortino and R. N. Pioli. 2020. Study of plant origin compounds with potential antifungal action against pathogens of cultivated plants. Bashir, L., A. Abdulkadir, M. Shah, A. Hamisu, U. Sharif and A. Kamalu. 2020. Phytochemical screening and antifungal potentials of Citrus limon peels against Fusarium oxysporum and Rhizopus stolonifer causing rots in water melon ( Citrullus lanatus L.). J. Exp. Sci. 11:1-5. Bihal, R., J. M. Al-Khayri, A. N. Banu, N. Kudesia, F. K. Ahmed, R. Sarkar, A. Arora and K. A. Abd-Elsalam. 2023. Entomopathogenic fungi: an eco-friendly synthesis of sustainable nanoparticles and their nanopesticide properties. Microorganisms 11:1617-1625. Chaubey, M. K. 2022. Chapter-3 Biology of Common Stored-Grain Insect Pests. Lat. Trends Zool. Entomol. Sci: 31. Cun, D., C. Zhang, H. Bera and M. Yang. 2021. Particle engineering principles and technologies for pharmaceutical biologics. Adv. Drug Deliv. Rev. 174:140-167. El-Gendy, R. M., E. M. Abd-ElAzeem and S. N. El-Shafiey. 2024. Moringa oleifera and Ruta angustifolia fixed oils and a prospective method to obstruct pupal development in cotton leafworm. J. Saudi Soc. Agric. Sci. 23:184-191. Ferreyra-Suarez, D., O. García-Depraect and R. Castro-Muñoz. 2024. A review on fungal-based biopesticides and biofertilizers production. Ecotoxicol. Environ. Saf. 283:116940-116945. Gadelhaq, S. M., S. M. Aboelhadid, A.-A. S. Abdel-Baki, K. M. Hassan, W. M. Arafa, S. M. Ibrahium, S. Al-Quraishy, A. O. Hassan and S. G. Abd El-Kareem. 2023. Safety and Efficacy of Pure and a Nanosuspension of D-limonene for Controlling Pigeon Lice. J. Med. Entomol. 60:148-158. Gupta, H., P. Bhatnagar and L. Shahani. 2024. Teratogenic effects of voriconazole (anti-fungal drug) on Swiss albino mice. Reprod. Toxicol. 129:108681-108686. Hasaballah, A., T. Selim, M. Tanani and E. Nasr. 2021. Lethality and vitality efficiency of different extracts of Salix safsaf leaves against the house fly, Musca domestica L.(Diptera: Muscidae). Afr. Entomol. 29:479-490. Hazafa, A., N. Jahan, M. A. Zia, K.-U. Rahman, M. Sagheer and M. Naeem. 2022. Evaluation and optimization of nanosuspensions of Chrysanthemum coronarium and Azadirachta indica using Response Surface Methodology for pest management. Chemosphere 292:133400-133411. Hou, T., S. S. Sana, H. Li, Y. Xing, A. Nanda, V. R. Netala and Z. Zhang. 2022. Essential oils and its antibacterial, antifungal and anti-oxidant activity applications: A review. Food Biosci. 47:101712-101716. Hussein, N. N., K. Al-Azawi, G. M. Sulaiman, S. Albukhaty, R. M. Al-Majeed, M. Jabir, A. G. Al-Dulimi, H. A. Mohammed, N. Akhtar and R. Alawaji. 2023. Silver-cored Ziziphus spina-christi extract-loaded antimicrobial nanosuspension: overcoming multidrug resistance. Nanomed. 18:1839-1854. Ibrahium, S. M., A. A. Wahba, A. A. Farghali, A.-A. S. Abdel-Baki, S. A. Mohamed, S. Al-Quraishy, A. O. Hassan and S. M. Aboelhadid. 2022. Acaricidal activity of tea tree and lemon oil nanoemulsions against Rhipicephalus annulatus. Pathogens 11:1506-1515. Iqbal, H., N. Jahan, S. Ali, A. Shahzad and R. Iqbal. 2024. Formulation of Moringa oleifera nanobiopesticides and their evaluation against Tribolium castaneum and Rhyzopertha dominica. J. Plant Dis. Protect. 131:133-142. Jampílek, J., K. Kráľová and P. Fedor. 2020. Bioactivity of nanoformulated synthetic and natural insecticides and their impact on environment. Nanopesticides: From Research and Development to Mechanisms of Action and Sustainable Use in Agriculture. Springer, New York City. pp.165-225. Karabulut, F., A. Khan, E. F. Abd_Allah, J. A. Parray and A. Hashem. 2024. Omics and other biotechnological tools for biopesticide and microbial bioremediation. In: Microbiome-Assisted Bioremediation. Elsevier, Netherlands. pp.103-128. Kocyigit, E., B. Kocaadam-Bozkurt, O. Bozkurt, D. Ağagündüz and R. Capasso. 2023. Plant toxic proteins: their biological activities, mechanism of action and removal strategies. Toxins 15:356-360. Laoudi, T., K. Khelfane-Goucem, S. Hamani-Aoudjit, F. Chebrouk, T. Amrouche, L. Saher and A. Kellouche. 2023. Chemical composition of essential oils from the leaves of Schinus molle and Cupressus sempervirens and their insecticidal activity against Oryzaephilus surinamensis (Coleoptera: Silvanidae). J. Essent. Oil Bear. Plants 26:309-322. Liu, C., J. Wang, W. Su, G. Chen and J. Zhang. 2024. Effects of mango and partridge tea extracts on microbial, physical, and chemical properties of tilapia fillets treated with in-package cold plasma during refrigerator storage. Food Qual. Saf. 8:fyae001-fyae006. Magi, M. S., L. Lopez-Vidal, M. C. García, C. C. Stempin, C. Marin, B. Maletto, S. D. Palma, J. P. Real and A. F. Jimenez-Kairuz. 2024. Organic solvent-free benznidazole nanosuspension as an approach to a novel pediatric formulation for Chagas disease. Ther. Deliv. 1:1-18. Marques, S. M. and L. Kumar. 2023. Factors affecting the preparation of nanocrystals: characterization, surface modifications and toxicity aspects. Expert Opin. Drug Deliv. 20:871-894. Miedaner, T. and M. M. Garbelotto. 2024. Human-mediated migration of plants, their pathogens and parasites. J. Plant Pathol. 1:1-25. Morteza-Semnani, K., M. Saeedi, J. Akbari, M. Moazeni, H. Seraj, E. Daftarifard, M. Tajbakhsh, S. M. H. Hashemi and A. Babaei. 2021. Fluconazole nanosuspension enhances in vitro antifungal activity against resistant strains of Candida albicans. Pharm. Sci. 28:112-129. Osdaghi, E., J. M. van der Wolf, H. Abachi, X. Li, S. H. De Boer and C. A. Ishimaru. 2022. Bacterial ring rot of potato caused by Clavibacter sepedonicus: a successful example of defeating the enemy under international regulations. Mol. Plant Pathol. 23:911-932. Palermo, D., G. Giunti, F. Laudani, V. Palmeri and O. Campolo. 2021. Essential oil-based nano-biopesticides: Formulation and bioactivity against the confused flour beetle Tribolium confusum. Sustainability 13:9738-9746. Papanikolaou, N. E., N. G. Kavallieratos, V. Iliopoulos, E. Evergetis, A. Skourti, E. P. Nika and S. A. Haroutounian. 2022. Essential oil coating: Mediterranean culinary plants as grain protectants against larvae and adults of Tribolium castaneum and Trogoderma granarium. Insects 13:160-165. Park, J.-S., J.-H. Choi, M.-Y. Joung, I.-G. Yang, Y.-S. Choi, M.-J. Kang and M.-J. Ho. 2024. Design of High-Payload Ascorbyl Palmitate Nanosuspensions for Enhanced Skin Delivery. Pharmaceutics 16:171-176. Perween, N., S. Alshehri, T. Easwari, V. Verma, M. Faiyazuddin, A. Alanazi and F. Shakeel. 2021. Investigating the feasibility of mefenamic acid nanosuspension for pediatric delivery: Preparation, characterization, and role of excipients. Processes 9:568-574. Porwal, O. 2022. Box-Behnken Design-based formulation optimization and characterization of spray dried rutin loaded nanosuspension: State of the art. S. Afr. J. Bot. 149:807-815. Rani, N., A. Duhan, A. Pal, P. Kumari, R. K. Beniwal, D. Verma, A. Goyat and R. Singh. 2023. Are nano-pesticides really meant for cleaner production? An overview on recent developments, benefits, environmental hazards and future prospectives. Journal of Cleaner Production, 411: 137232. Rauf, S. 2024. Insecticide resistance in agricultural pests: challenges and solutions. Trends Biotech. Plant Sci. 2:36-41. Raveau, R., J. Fontaine and A. Lounès-Hadj Sahraoui. 2020. Essential oils as potential alternative biocontrol products against plant pathogens and weeds: A review. Foods 9:345-365. Rennick, B. 2023. Truffles in Michigan: Impacts of Herbicides on Their Growth, Effectiveness of In-Field Inoculations, and the Discovery of a Local Truffle (Tuber rugosum). Michigan State University, USA. pp.15-20. Sánchez-Hernández, E., V. González-García, A. Palacio-Bielsa, B. Lorenzo-Vidal, L. Buzón-Durán, J. Martín-Gil and P. Martín-Ramos. 2023. Antibacterial activity of Ginkgo biloba extracts against Clavibacter michiganensis subsp. michiganensis, Pseudomonas spp., and Xanthomonas vesicatoria. Horticulturae 9:455-461. Singh, S., S. Sharma and A. K. Dubey. 2024. Enhanced Antibacterial and Antibiofilm Activities of Actinobacterial Therapeutic Metabolites Mediated Nanosuspension. Part. Syst. Charact. 1:2300196-2300200.. Summer, M., H. M. Tahir, S. Ali, S. Nawaz, R. Abaidullah, S. Mumtaz, A. Ali and A. H. Gormani. 2024. Nanobiopesticides as an Alternative and Sustainable Solution to Tackle Pest Outbreaks. J. Kans. Entomol. Soc. 96:112-136. Van Driessche, A. E., W. L. Ling, G. Schoehn and M. Sleutel. 2022. Nucleation of glucose isomerase protein crystals in a nonclassical disguise: The role of crystalline precursors. Proc. Natl Acad. Sci.119:e2108674110-e2108674119. Visakh, N. U., B. Pathrose, M. Chellappan, M. Ranjith, P. Sindhu and D. Mathew. 2022. Chemical characterisation, insecticidal and antioxidant activities of essential oils from four Citrus spp. fruit peel waste. Food Biosci. 50:102158-102163. Vishnu, M., K. Malaisamy, R. Soundararajan, A. Suganthi, A. Subramanian, M. Senthilkumar, K. Rameash, K. Madesh and G. Kasivelu. 2024. Nano-bioformulations: emerging trends and potential applications in next generation crop protection. Environ. Sci. Nano. 1:75-95. Wang, W., Y. Weng, T. Luo, Q. Wang, G. Yang and Y. Jin. 2023. Antimicrobial and the resistances in the environment: ecological and health risks, influencing factors, and mitigation strategies. Toxics 11:185-190. Wu, S., Y. Sun, W. Jia, F. Wang, S. Lu and H. Zhao. 2023. Estimation of Above-Ground Carbon Storage and Light Saturation Value in Northeastern China’s Natural Forests Using Different Spatial Regression Models. Forests 14:1970-1986. Zaman, N., M. Rizwan, A. Iqbal, A. Rauf, Y. S. Al-Awthan and O. Bahattab. 2024. Phytochemistry and Medicinal Uses of the Family Rutaceae. In: Phytochemical and Pharmacological Investigation of the Family Rutaceae. Apple Academic Press, Canada. pp.51-69. Zhou, X., L. Lei, Y. Zeng, X. Lu, F. Liang, L. Zhang and G. Lin. 2023. High salinity effects on the depletion attraction in colloid-polymer mixtures. J. Colloid Interface Sci. 631:155-164. Supplementary Files floatimage1.jpeg Graphical Abstract Supplementaryfiles.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6714957","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":481792056,"identity":"1d76f640-6649-440b-8701-16432b00c9f2","order_by":0,"name":"Rabia bibi","email":"","orcid":"","institution":"University of Agriculture Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Rabia","middleName":"","lastName":"bibi","suffix":""},{"id":481792057,"identity":"7aeb097d-a9b6-471d-8654-783e1def4f31","order_by":1,"name":"Nazish Jahan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYJCCA4wNbAwGDAyMDxgY5ID8BJAYcVqYgdiYOC0MjA0MIC1sEkRpMWc//vDgzx188ubsZ49V89QYMPCz5xgwF5zBrcWyJyHhMO8ZNsOdPXlpt3mOGTBI9rwxYJ5xA7cWgwMJBw4ztrExbjiQY3abh+0Pg8ENoC08H/BoOf+w4eDPNjb7DeffmBXz/DNgsCeo5UYywwHeNrbEDTdyzJh52wwYDCRAWvA57MYzhsNALckbbrwxlpzbZ8AjceZZweEZeLxvcD798cefbcdsN5zPMfzw5puBHH978sbHBcdwa4EChAoeEHGYoAYGhhpULjMRWkbBKBgFo2DkAACBRVd6qde/0gAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0009-0008-6549-8694","institution":"University of Agriculture Faisalabad Faculty of Sciences","correspondingAuthor":true,"prefix":"","firstName":"Nazish","middleName":"","lastName":"Jahan","suffix":""},{"id":481792058,"identity":"605d256e-55eb-41e4-a8d9-db7ea69a1910","order_by":2,"name":"Kousar Rasheed","email":"","orcid":"","institution":"University of Agriculture Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Kousar","middleName":"","lastName":"Rasheed","suffix":""},{"id":481792059,"identity":"4f6c881e-4480-4b57-8e35-a02d34196bde","order_by":3,"name":"Samiah Samiah","email":"","orcid":"","institution":"University of Agriculture Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Samiah","middleName":"","lastName":"Samiah","suffix":""},{"id":481792060,"identity":"fa5c760a-2cf6-49dd-805b-18723bdbb242","order_by":4,"name":"Aqsa Hameed","email":"","orcid":"","institution":"University of Agriculture Faisalabad","correspondingAuthor":false,"prefix":"","firstName":"Aqsa","middleName":"","lastName":"Hameed","suffix":""}],"badges":[],"createdAt":"2025-05-21 09:37:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6714957/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6714957/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86350025,"identity":"9a3a9b61-ab5b-4fe8-8895-2502951b28ec","added_by":"auto","created_at":"2025-07-09 15:47:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":146960,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size and PDI value of \u003cem\u003eC. limon\u003c/em\u003e nano suspension with Tween 80 stabilizer (Akhavan-Mahdavi\u003cem\u003e et al.\u003c/em\u003e, 2022)\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/b74c46e35d12d203d3e200e7.png"},{"id":86349467,"identity":"2c2564a1-ce09-419d-94e5-69d571470d22","added_by":"auto","created_at":"2025-07-09 15:39:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":87415,"visible":true,"origin":"","legend":"\u003cp\u003e3D graph represents the synergetic effect of amount of plant extract and conc. of stabilizer on particle size of \u003cem\u003eC. limon\u003c/em\u003e nanosuspension (Hussein\u003cem\u003e et al.\u003c/em\u003e, 2023)\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/a255109ed2814ee22e139a17.png"},{"id":86349448,"identity":"dd1c9c48-f5b7-4745-bc9a-04688a3b4051","added_by":"auto","created_at":"2025-07-09 15:39:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82111,"visible":true,"origin":"","legend":"\u003cp\u003e3D graph represents the synergetic effect of amount of plant extract and conc. of stabilizer on PDI of \u003cem\u003eC. limon\u003c/em\u003e nanosuspension (Hazafa\u003cem\u003e et al.\u003c/em\u003e, 2022)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/a22afea7d770019c50100cdb.png"},{"id":86350023,"identity":"c3486c64-f532-4b7b-baf6-ed9f87ce7f58","added_by":"auto","created_at":"2025-07-09 15:47:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":143431,"visible":true,"origin":"","legend":"\u003cp\u003ePesticidal activity of \u003cem\u003eC. limon \u003c/em\u003epeels coarse extract against\u003cem\u003e T. castaneum \u003c/em\u003e(Visakh et al., 2022)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/2315a103c1b361a61c3199b6.png"},{"id":86349461,"identity":"e2a9780b-dd12-4d7f-a8f0-be24904ae85b","added_by":"auto","created_at":"2025-07-09 15:39:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":154327,"visible":true,"origin":"","legend":"\u003cp\u003ePesticidal activity of \u003cem\u003eC. limon\u003c/em\u003e nanosuspension against \u003cem\u003eT. castaneum \u003c/em\u003e(Iqbal et al., 2024)\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/b606d5724efa22ac67b16b5a.png"},{"id":86350022,"identity":"3bca4cbe-d534-404f-a449-3804b2827a1a","added_by":"auto","created_at":"2025-07-09 15:47:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":157549,"visible":true,"origin":"","legend":"\u003cp\u003ePesticidal activity of Malathion (control) against\u003cem\u003e T. castaneum \u003c/em\u003e(Elmadawy et al., 2024)\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/a4fbfa4d642fc35da5e90cf2.png"},{"id":86349460,"identity":"31882f19-a7ac-42d7-9f6a-26c887b23396","added_by":"auto","created_at":"2025-07-09 15:39:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":106817,"visible":true,"origin":"","legend":"\u003cp\u003ePesticidal activity of \u003cem\u003eC. limon\u003c/em\u003e peels coarse extract against \u003cem\u003eO.surinamensis \u003c/em\u003e(Babarinde \u003cem\u003eet al\u003c/em\u003e., 2021)\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/194fc5159a3195c81b173ddb.png"},{"id":86349433,"identity":"a17a3727-60d7-43d8-b1c7-b016cf4d32a9","added_by":"auto","created_at":"2025-07-09 15:39:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":112805,"visible":true,"origin":"","legend":"\u003cp\u003ePesticidal activity of \u003cem\u003eC. limon\u003c/em\u003e nano suspension against \u003cem\u003eO.surinamensis \u003c/em\u003e(Laoudi \u003cem\u003eet al\u003c/em\u003e., 2023)\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/53b67e03bef7622a4cc69845.png"},{"id":86349514,"identity":"bc0ecc81-5805-4d76-8455-e18ee929739c","added_by":"auto","created_at":"2025-07-09 15:39:09","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":122741,"visible":true,"origin":"","legend":"\u003cp\u003ePesticidal activity of Malathion (control) against \u003cem\u003eO.surinamensis \u003c/em\u003e(Laoudi \u003cem\u003eet al\u003c/em\u003e., 2023)\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/c221647954ff0006163128f7.png"},{"id":86349434,"identity":"11401b28-978f-41e2-9465-e15bf0693a28","added_by":"auto","created_at":"2025-07-09 15:39:04","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":108786,"visible":true,"origin":"","legend":"\u003cp\u003ePesticidal activity of \u003cem\u003eC. limon \u003c/em\u003epeels coarse extract against \u003cem\u003eS. zeamais \u003c/em\u003e(Laoudi \u003cem\u003eet al\u003c/em\u003e., 2023)\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/8abfd6e7093cbce130e3c111.png"},{"id":86349432,"identity":"fe58694c-3086-4f1b-a0ac-fa119e99de31","added_by":"auto","created_at":"2025-07-09 15:39:04","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":111086,"visible":true,"origin":"","legend":"\u003cp\u003ePesticidal activity of \u003cem\u003eCitrus limon\u003c/em\u003e nanosuspension against \u003cem\u003eS. zeamais\u003c/em\u003e (Iqbal\u003cem\u003e et al.\u003c/em\u003e, 2024)\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/d7f82a5d3a1970f2414cff25.png"},{"id":86349463,"identity":"1e603492-7f95-4172-8a76-89c83f0ef2d3","added_by":"auto","created_at":"2025-07-09 15:39:07","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":106427,"visible":true,"origin":"","legend":"\u003cp\u003ePesticidal activity of Malathion (control) against \u003cem\u003eS. zeamais\u003c/em\u003e (Aisvarya\u003cem\u003e et al.\u003c/em\u003e, 2024)\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/6691cf74d018d7f8f31556e8.png"},{"id":86349492,"identity":"757cd82e-ead4-4c5f-a388-f17869d1d0bd","added_by":"auto","created_at":"2025-07-09 15:39:09","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":148954,"visible":true,"origin":"","legend":"\u003cp\u003eZone of inhibition for \u003cem\u003eC. limon\u003c/em\u003e peels extract against \u003cem\u003eP. syringae\u003c/em\u003e and \u003cem\u003eC. michiganesis\u003c/em\u003e (Arredondo-Valdés\u003cem\u003e et al.\u003c/em\u003e, 2020)\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/fc970034a4280dc5d02d7dc1.png"},{"id":86349484,"identity":"594b747e-9070-4a77-81e6-9c859e6895f6","added_by":"auto","created_at":"2025-07-09 15:39:09","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":137221,"visible":true,"origin":"","legend":"\u003cp\u003eZone of inhibition for \u003cem\u003eC. limon\u003c/em\u003e nanaosuspension against \u003cem\u003eP. syringae\u003c/em\u003e and \u003cem\u003eC. michiganesis\u003c/em\u003e (Almahdy\u003cem\u003e et al.\u003c/em\u003e, 2024)\u003c/p\u003e","description":"","filename":"floatimage16.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/f402551ed34f956a181fabc9.png"},{"id":86349468,"identity":"bf984669-7c0a-41c7-9fe3-a43f8803fefb","added_by":"auto","created_at":"2025-07-09 15:39:08","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":155598,"visible":true,"origin":"","legend":"\u003cp\u003eZone of inhibition for Ciprofloxacin (control) against \u003cem\u003eP. syringae\u003c/em\u003e and \u003cem\u003eC. michiganesis\u003c/em\u003e (Sánchez-Hernández\u003cem\u003e et al.\u003c/em\u003e, 2023)\u003c/p\u003e","description":"","filename":"floatimage17.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/db51c600cce3b5680beaa5bf.png"},{"id":86349438,"identity":"4c03a34b-116e-4e1e-990d-9f97ce76d63a","added_by":"auto","created_at":"2025-07-09 15:39:05","extension":"png","order_by":16,"title":"Figure 16","display":"","copyAsset":false,"role":"figure","size":121648,"visible":true,"origin":"","legend":"\u003cp\u003eZone of inhibition of \u003cem\u003eC. limon\u003c/em\u003e peels extract against \u003cem\u003eF. oxysporum\u003c/em\u003e and \u003cem\u003eR. stolonifer\u003c/em\u003e (Bashir\u003cem\u003e et al.\u003c/em\u003e, 2020)\u003c/p\u003e","description":"","filename":"floatimage18.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/f9fbb5b42cfa25333000930b.png"},{"id":86349427,"identity":"8bec9c0b-c695-4f56-998e-fe512e4a303d","added_by":"auto","created_at":"2025-07-09 15:39:02","extension":"png","order_by":17,"title":"Figure 17","display":"","copyAsset":false,"role":"figure","size":123544,"visible":true,"origin":"","legend":"\u003cp\u003eZone of inhibition of \u003cem\u003eC. limon \u003c/em\u003enanosuspension against \u003cem\u003eF. oxysporum\u003c/em\u003e and \u003cem\u003eR. stolonifer\u003c/em\u003e (Raveau\u003cem\u003e et al.\u003c/em\u003e, 2020)\u003c/p\u003e","description":"","filename":"floatimage19.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/a91953d3c6836576d27f1630.png"},{"id":86349472,"identity":"f75bdfc2-f2f8-40c1-a646-1079a240f847","added_by":"auto","created_at":"2025-07-09 15:39:08","extension":"png","order_by":18,"title":"Figure 18","display":"","copyAsset":false,"role":"figure","size":109749,"visible":true,"origin":"","legend":"\u003cp\u003eZone of inhibition of Iitraconazole (control) against\u003cem\u003e F. oxysporum\u003c/em\u003e and \u003cem\u003eR. stolonifer \u003c/em\u003e(Elmadawy\u003cem\u003e et al.\u003c/em\u003e, 2024)\u003c/p\u003e","description":"","filename":"floatimage20.png","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/a9153220ab07a4bc701afd82.png"},{"id":99308015,"identity":"a42d3e1e-c854-4a85-b4d8-c1ae19c50260","added_by":"auto","created_at":"2025-12-31 16:07:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4495490,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/821d9567-5cec-44d9-993e-ab7bf2a2eb46.pdf"},{"id":86349426,"identity":"b61b30c8-ca37-4707-a3b6-ddcf7eb4404c","added_by":"auto","created_at":"2025-07-09 15:39:02","extension":"jpeg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":214048,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/91cec623de9c37f36713d895.jpeg"},{"id":86350030,"identity":"a39d7798-db1d-4bfa-9b40-f055e523c38b","added_by":"auto","created_at":"2025-07-09 15:47:09","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1194316,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfiles.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6714957/v1/37529975616b63895da93f74.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\u003cem\u003eC. limon\u003c/em\u003e Peels Based Nano-Bio pesticides: Formulation and Bioactivity against the Stored Pests and Microbes\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003ePulses and cereals are attacked in stores by various insect pests. The storage insects are actually a major threat to storage grains and they lead to direct and indirect losses of cereal grains in storage (DEMIS and YENEWA, 2022). Since the beginning of agriculture and settled communities, grains have been the primary source of food for humans (Miedaner and Garbelotto, 2024). Some of the storage insect pests which attack storage grains are rice weevils, maize weevil, lesser grain borer, granary, weevil, red flour beetle, khapra beetle, and pulse beetles. In order to meet their needs for food and shelter, stored grain pests infest grains during storage, which results in both quantitative and qualitative losses (Chaubey, 2022).\u003c/p\u003e\u003cp\u003eThe indiscriminate usage of traditional pesticides has resulted in pesticide resistance, which also has an impact on soil fertility. Ultimately, there is a need to find alternative means for managing pest infestation (Summer \u003cem\u003eet al.\u003c/em\u003e, 2024). Natural compounds and microorganisms have also been utilized as bio pesticides all over the world (Assadpour \u003cem\u003eet al.\u003c/em\u003e, 2024). Despite, their progress have been increasing in recent decades, but bio pesticides use remains limited due to a lack of comprehensive knowledge of their modes of action, suitable storage and handling (Karabulut \u003cem\u003eet al.\u003c/em\u003e, 2024).\u003c/p\u003e\u003cp\u003eOther reasons for not considering bio pesticides as mainstream pest management tools include their relatively higher cost, lower stability, and reduced efficacy (Ferreyra-Suarez \u003cem\u003eet al.\u003c/em\u003e, 2024). The research on sustainable and effective pest management has led the scientists to look for the innovative material solutions. Biodegradable and biocompatible substances with intelligent properties have the potential for developing safer pesticides (Abdollahdokht \u003cem\u003eet al.\u003c/em\u003e, 2022). Nanotechnology offers a transformative approach by enabling the creation of precisely engineered pesticide delivery systems. These Nano-pesticides can be designed to release the active ingredients in a controlled (Vishnu \u003cem\u003eet al.\u003c/em\u003e, 2024). Lemon is a valuable medicinal plant species of the Rutaceae family. Lemon is grown primarily for its alkaloids, which have anticancer properties and the bactericidal properties of crude extracts in different sections of the plant (such as the peels, flowers, stems, leaves, and roots) against clinically important bacterial strain (Zaman \u003cem\u003eet al.\u003c/em\u003e, 2024). \u003cem\u003eCitrus limon\u003c/em\u003e peels have antibacterial, antifungal, and antimicrobial properties. However, there are several limitations regarding the use of \u003cem\u003eC. limon\u003c/em\u003e peels as bio pesticides, such as low solubility, instability, poor bioavailability, and slow release of active ingredients.\u003c/p\u003e\u003cp\u003eThe purpose of our present study is to formulate a \u003cem\u003eC. limon\u003c/em\u003e nanosuspension to enhance the dissolution rate, control the release of active ingredients, and improve stability and bioavailability to increase its therapeutic properties against agricultural pests as a nano-biopesticide. After synthesis, the insecticidal activity of this nanosuspension was examined against economically important stored grain pests such as \u003cem\u003eTribolium confusum\u003c/em\u003e, \u003cem\u003eOryzaephilus surinamensis\u003c/em\u003e, and \u003cem\u003eSitophilus zeamais\u003c/em\u003e. The objectives of this study were, firstly, the formulation and optimization of an eco-friendly nanosuspension of \u003cem\u003eCitrus limon\u003c/em\u003e peels with increased pest control efficacy, and secondly, the evaluation of the pesticidal and antimicrobial potential of the prepared nanosuspension. Results from laboratory trials showed the excellent efficacy of the \u003cem\u003eC. limon\u003c/em\u003e nanosuspension and its promising insecticidal activity against the phyto-pathogenic species and microbes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Chemicals\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eCitrus limon\u003c/em\u003e peels were sourced from a local market in Faisalabad, Pakistan. The following chemicals were procured for the study: ethanol (99.5% purity), polyvinyl pyrrolidone (PVP; 99% purity), poly vinyl alcohol (PVA; 99% hydrolyzed), hydroxyl propyl methylcellulose (HPMC; 99% purity), Tween 80 (99% purity), and polyethylene glycol (PEG; 99.5% purity) from Sigma-Aldrich (USA), and nutrient agar from Merck (Germany).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e2.2 Collection of sample\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eCitrus limon\u003c/em\u003e peels were used for the effective formation of the nano suspension. Lemon peels (\u003cem\u003eC. limon\u003c/em\u003e) were collected from a local market in Faisalabad and cleaned to remove dirt and dust particles. The lemon peels were ground into a fine powder and stored in an airtight jar for subsequent studies.\u003c/p\u003e\n\u003ch3\u003e2.3 Preparation of plant extract\u003c/h3\u003e\n\u003cp\u003eThe dried plant sample was crushed into a fine powder using a Binatone MX10 blender. All compounds were extracted from the plants using a Soxhlet apparatus. To separate the desirable compounds from \u003cem\u003eC. limon\u003c/em\u003e, ethanol was used as a solvent. The extraction process lasted for 6 to 8 hours, or until the intense yellow-green color of the lemon peels within the thimble became colorless. The extract of lemon peels was then filtered and concentrated for 2 to 3 hours using a rotary evaporator (Buchi, CH-9230 Flawil 1, Switzerland). After this, the concentrated extract was stored in the refrigerator (2 to 8\u0026deg;C) for further analysis (Liu \u003cem\u003eet al.\u003c/em\u003e, 2024).\u003c/p\u003e\n\u003ch3\u003e2.4 Formulation of nanosuspension\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4.1 Screening of stabilizers\u003c/h2\u003e\n \u003cp\u003eThe nanosuspension formulation included six distinct stabilizers: polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), sodium lauryl sulphate (SLS), Tween 80, hydroxyl propyl methyl cellulose (HPMC), and polyethylene glycol (PEG) (Park \u003cem\u003eet al.\u003c/em\u003e, 2024). Throughout experiment, the solvent (ethanol) to anti-solvent (distilled water) ratio was kept at 10:20. An amount of 0.25 g of the each stabilizer was measured using a weighing balance and dispersed in 100 ml of water (anti-solvent) in 500 ml Pyrex flask. After that, 0.25 g of each extract of plant was added in 10 ml of suitable solvent (ethanol) and properly mixed. The mechanical shaker (RW 28 electronic overhead rotator, Thomas Research) was used for 6 hours around 4000 rpm. After stirring the mixture, each nano suspension was stored at room temperature (27\u0026thinsp;\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e\u0026plusmn;\u003c/span\u003e\u0026thinsp;3\u0026deg;C) or in the refrigerated (8\u0026ndash;10\u0026deg;C) for one week to find the best stabilizer (Magi \u003cem\u003eet al.\u003c/em\u003e, 2024).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4.2 Optimization of formulation parameters\u003c/h2\u003e\n \u003cp\u003eAfter stabilizers screening, different amounts of stabilizers and plant extract were optimized to formulate an effective nano suspension with minimum particle size. For all complete runs, a solvent to anti-solvent ratios (S/AS) were kept constant 10:20. Three various parameters including plant extract and amounts of stabilizers (0.25-1) as well as solvent to anti-solvent ratio (10\u0026ndash;20) were optimized by Design Expert (Ver. 13) software along with two responses (particles size and PDI). The nano-precipitation method (bottom up approach) was used to produce \u003cem\u003eC. limon\u003c/em\u003e nanosuspension (Cun \u003cem\u003eet al.\u003c/em\u003e, 2021). The central composite design of RSM provided an experimental design for optimizing various parameters.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003e2.5 Characterization of nanosuspension\u003c/h3\u003e\n\u003cp\u003eNanosuspension was characterized by employing following technique.\u003c/p\u003e\n\u003ch3\u003e2.5.1 Particle size analysis\u003c/h3\u003e\n\u003cp\u003eThe PDI (polydispersity index) and average particle size (Z-average-nm) of the synthesized \u003cem\u003eC. limon\u003c/em\u003e nanosuspension were evaluated using a DLS (Dynamic Light Scattering 0012), a 12 mm cell through Zeta Sizer. The plant sample was taken in glass cuvettes. For each RSM prepared nanosuspension of \u003cem\u003eC. limon\u003c/em\u003e peels, histogram was recorded (Perween \u003cem\u003eet al.\u003c/em\u003e, 2021).\u003c/p\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5.2 Stability studies\u003c/h2\u003e\n \u003cp\u003eTo check the physical stability, \u003cem\u003eC. limon\u003c/em\u003e nanosuspension was placed in refrigerator at 4\u003csup\u003eo\u003c/sup\u003eC or at normal room conditions (25\u003csup\u003eo\u003c/sup\u003eC) for 3 months. Less than 0.5 PDI and 100\u0026ndash;600 nm particle size value proved the stability of nanosuspension.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e2.6 Anti-microbial activity\u003c/h2\u003e\n \u003cp\u003eAntimicrobial activity of \u003cem\u003eC. limon\u003c/em\u003e nanosuspension was investigated by well diffusion method. Two bacterial strains (gram positive \u003cem\u003eClavibacter michiganesis\u003c/em\u003e and gram negative \u003cem\u003ePseudomonas syringae\u003c/em\u003e) were selected to analyze anti-bacterial activity of \u003cem\u003eC. limon\u003c/em\u003e extract and its optimized nano suspension. 9.2 g (2.3 g in 100 ml) nutrient agar was dissolved in 400 ml distilled water. For sterilization, flask holding media was wrapped in aluminium foil and put in autoclave for 20 min at 121\u0026deg;C. This media was distributed evenly in petri-dishes (2\u0026ndash;3 mm) in form of thin film gel. Wells were produced on agar medium, and 2ul of each bacterial strain was injected into different sterilised plates. At 25\u0026deg;C, every plate was incubated for 24 hours and zones of inhibition were measured (Anbazhagan, 2024).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7 Pesticidal activity\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eC. limon\u003c/em\u003e peels extract was used in five different concentrations (50, 100 150, 200 and 250 mg/ml) along with control against three insects (red flour beetle, corn weevil and saw toothed beetle). Extracts of plant were also synthesized individually at very same concentration in distilled water in order to compare with nano suspension (Tran\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, 2024). Simply, plastic petri dishes were utilized as enclosures for insects over Whatman No. 1 filter paper. 1ml of each plant extract, its nano suspension and control was sprinkled over each Whatman No. 1 filter paper and allowed to dry for 2 minutes under a fume hood. Twenty adult beetles of each pest were introduced into every petri dish and tightly wrapped using plastic tape. The petri dishes were then placed in an incubator. After 2, 48 and 72 hours, the percentage of insects that died was calculated (Grigg\u003cem\u003e\u0026nbsp;et al.\u003c/em\u003e, 2024).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e2.7.1 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eThe efficacy of \u003cem\u003eC. limon\u003c/em\u003e peels nano suspension and extract dilutions was determined using analysis of variance (ANOVA). All the trials were repeated three times, and Mortality rate of insects was calculated by Abbot\u0026apos;s formula (Hasaballah \u003cem\u003eet al.\u003c/em\u003e, 2021).\u003c/p\u003e\n \u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAANIAAAAsCAYAAAAQAeYrAAAAAXNSR0IArs4c6QAAAARnQU1BAACxjwv8YQUAAAAJcEhZcwAADsMAAA7DAcdvqGQAAAnWSURBVHhe7ZzLy01fGMeX39x9JkmYCCm3JAyU69TlFSVFRFKSSygD91tKcinKyCUGJu5lgIHcohQTkmTk7g94f/uzzv6+Hsve++xz3uM973usT632Xmuv297nedZ69rPWPr3aE1wkEukU/6XHSCTSCaIiRSINICpSJNIAoiJFIg0gKlIk0gCiIkUiDSAqUiTSAKIiNZHt27e7ixcvprFykP/gwYPu8+fPaUqkW8CCbKTraWtra3/+/Hkaa2+fPXs2C+Pt27ZtS1MqkO/NmzdprALlyC/u37/vyxIuXLiQplY4depUxzXx6dOn9jVr1rT379/fp3MMyxWh+qjDQr90jT6Ja9eutU+YMKGjLe6RPrQSUZGaAEJrhRBBkwIhcBIy8h04cMCfh1DeCj/lKUuwSIBpQ6CEpEtBaSOvnSysYliFkHLawUBKLsXiSJ5QCXs60bRrAlevXnVLlixJY869fPnSzZ07158PHDjQvXr1yptu586dcytWrPDpIZSnHtG3b183ceJE9+TJE/f27Vufdv36dTdr1ix/3qdPH3988eKFu3Xrlrt06ZIbNmyYT9u8ebMPZaEu+jl8+HB3584dn0Z/Hz9+7CZNmuT7Io4fP+4SpXFTp071cY779u1zJ0+e9PFWISpSE0DYR44cmcacGzJkiLtx44Y/RyARtrNnz7rly5d7gc2C8tRjGTp0qEtMQV8Wjh079ociPnz40B+lRJ1h06ZN7siRI/6cNjdu3OjPLbdv3/b9sowaNcofHzx44I+tQFSkJpCYVL8pyMyZM73A9erVyy1cuNDPKHfv3vXpzDKknz59Os1dgfLUE7Js2TI/2lMHyhIqzPfv39OzbHBk0F5WCAV/wYIFXpmZ5ejv4sWL0yu/+Pr1a3r2J717907Pej5RkZoAJpH1uqEUmEWJqe1NrOQdwyXvLO7KlStu5cqVLnkPcYcOHUpzV6A89YTMmzfPDRgwwJt01nwUU6ZM8UeZfyG0Tz+ygswzQb8x2+bPn+9mzJiRpv5O8j7m3r17l8YqfPjwwSXvSW7s2LFpSs+npRRJo6kFdzFpeYLTDJIXdf8elAWjPoqAkDF7YAYhsCNGjEhzVKA89QjyarZZtWqVz4/gS2F//Pjhj6ShgCirrvHcCGWhLpVFWb98+dJhQpJuZz3MU2ZIzajMXjt27HBbt2718ZYhGWlywbNCFoUirAuWYN2f9VKLZwevU5l+AnlDl3JXEnrtLHjU5Amjn7ivieMls1BeXjv77MPnLre6fS5ynys9UazfPIDVULnQ02d/A9sP7oE21FZYzmJlzj4jlcfjZ5cNQPeTJ3M8P1svSwrW2yjwRtp+1vRM0mMh6kBRxfph8gSkHqivFvRDVoOH1ExFAp5XKBA8Xytk9FE/rH32EpxWBUHnnu1vxP0zmFgF4DxPeS08K5XVMw0HJg1Ges6q1y4bFFFKUmlYIQs6QWe53ihF0ghSC2UUSXmarUgSgqLBKQspW9aI2ipwb8w8KJTiyFY48CAjCLqUIEuRKBMqBM8wzE9boXLRZtkBq7QiqfGsH54bolN5ikQ6ZQmcW1SvHX3pvPIrDuTJShdZikR/1KaUU4F025atT2lZ9xv5+2hHBnLFAi+/bR5FipQlE5IjW2cYB8lLGUorko46F1IA4BgqEmm2g8TVOSkRASFWHLIeAHHVpbz24YVl9CBsn63iCtq2eYB685RI7RSF8DlEaoffhZkpnClCihRJZmIIaZIlzVqKi1CeiqjJa7d7926/dmE3Wh4+fNinZ0E+8ttVc1brAS8R6w6JsPo43h3iSZ98PA8WL4EVdMCVmseJEye8e7Ya5Avv6/z585nrIlDkIlagzhC7JhNDJRSxfv16vw7F2lq9fPv2LT3L5+fPn+lZ/dSkSAhWMnJ79yXgUmYhMU/g7t2798dah9yv4dqCFKMIBJS2UMKw3s7AomUy+nUo+d8iVLYYigdNdmbwu2zZssW7zbszNa8j2VmJBUPWLLoKu05EHxoJgwN70BgcuLfp06enV/5E/SgKa9euTXNH6oF1p379+rmbN2/69TIWpusha6FYa4raExguNAsGexaOy1CzItlZiZGiaLMje6wQ+HBrCWlFgpoFddAeZYvarBfNlJiqzEx5syzUa9pFyoGgs5Nj7969Pn706FG/FamWRWMxevRof2QDr3j9+rU/Tp482R8BZb18+XIaq8BuE236rUZdOxs0KyUvY2lKNggcwjlt2rQ0pTKak1YkqKB3IR4qo7vehT5+/OiPjx498sdaGTx4sD9SD33R6ATMrqzCN2JDZ6Q+2BnR1tbmg34HBjni+/fvzzTxtGtDRwtbppC3nTt3+roJnGMy2i1KbLhFWfWezJH4unXrfLwqyeiZi7xeCtarkXQuPcv2YlkPCnmVjidGhOVCr4vS5T2z9di+cR72NVF035ZNE6rH3o8gPcv7E+ka8NLxG1j5wqumdILWk5CL8DcmHnpbyW/zIStZ63DIg22/7GIsxL8sDpgzZ463y1sVPm8fM2ZMVYvAwuj8/v17v5/O7lqP/KIu065VwczrCrNO5mqWzY/QsmmVEF4vulYGlGfRokUdSkQdOEYYPLQJFUi37xTkT0Z0t3Tp0jSlQp5jBfNL1+z7MfWymVbXUOqWwc9LEQ9T/t826zA7WKnH1AhNS5kwmKVaJJQZU3StDLTL/QlMGy100h+ZMTKJs6C8NZsoQx30xZpK5KOv1CtkNun5csxrpyfyz89IjPIaIfEk5rlCGwWj+549ezLdsrjf8RIxK/IizCxAWrVrZQg/b+czDC104gbmc3fg84pE6P15SPh5ey2fnOMowJOp58uxlUzof16REOxkQPFBJk+zePbsmRs3blwac278+PHpWfG1MuCBsp+3Dxo0qMPdy/dDfPBnv4XKIuvzdqj2yTmmHjsU5DFtReI7Ujci3M5iR/Sia3rXyQp6R2G5wjoKmNn0GfvTp0/9DLFhwwa3a9cu/85DejiwUJ56Qqp9ct6ILTjdnahI3QjMPWYegVCKomtFC8QypTC/rEMBMLXIg4nFTgJ2D7BexxevpKO8dt2G8tQTgoIl70W5n5yrD0X7Ins6UZGaBIpgFQMwr9i7iPASWHRmtK92rQys3Od93o6CnDlzxq1evdrPHjIhUQo7m4Sft9fyyTmKxm4YLYAzU+ItbBmSkSfSheCt4rHbYD2FeLpIw8MVLiwWXatG6LWzUK/1pvHpAXC0nsHQa6f+h95H4rqmevHqUV7poVevpxMV6R8Cd3PoMs9ydxNH2K3iUS7MF/lF3NnwD4G5hVeNfYy1eCjjzobqREWKRBpAdDZEIg0gKlIk0gCiIkUiDSAqUiTSAKIiRSKdxrn/AU5ijf+G8CsbAAAAAElFTkSuQmCC\"\u003e\u003c/p\u003e\n \u003cp\u003eWhere, MC\u0026thinsp;=\u0026thinsp;control mortality and MO\u0026thinsp;=\u0026thinsp;observed mortality\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e2.8 Anti-fungal activity\u003c/h2\u003e\n \u003cp\u003e16.8 g (4.2 g in 100 ml) of Potato Detrox Agar (PDA) was autoclaved in 400 ml of distilled water. For sterilization, the flask holding the media was wrapped in aluminium foil and put in autoclave for 20 min at 121\u0026deg;C (Rennick, 2023). The PDA medium was then put into the sterilized petri plates. In 1 ml of distilled water, two fungal strains (\u003cem\u003eF. oxysporum\u003c/em\u003e and \u003cem\u003eR. stolonifer\u003c/em\u003e) were added separately. PDA medium had already been poured into these sterile petri dishes. Wells were produced. The extract and its optimized nano suspension of various concentrations (150, 200, and 250 mg/ml) were poured into these wells. At 25\u0026deg;C, these petri dishes were incubated for 48 hours and zones of inhibition were measured after incubation (Morteza-Semnani \u003cem\u003eet al.\u003c/em\u003e, 2021).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eFor optimization studies, a central composite design using response surface methodology (RSM) was employed. The statistical experimental design was generated and analyzed using Design-Expert Software (Version 13). Analysis of variance was conducted with Tukey\u0026apos;s test, applying a significance level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Agbangba \u003cem\u003eet al.\u003c/em\u003e, 2024).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"RESULTS AND DISCUSSION","content":"\u003cdiv id=\"Sec18\"\u003e\n \u003ch2\u003e3.1 Screening of stabilizers\u003c/h2\u003e\n \u003cp\u003eSix distinct Food, Drug Administration approved and secure excipients, including SLS, Tween-80, PEG, PVP, PVA and HPMC were used as a stabilizer for synthesis of minimum sized \u003cem\u003eC. limon\u003c/em\u003e nano suspension. For suitable stabilizer selection, 0.25 g plant extract was added in 0.25 g stabilizer at fixed solvent to anti-solvent ratio (1:10). For further experimentation, a stabilizer with the lowest particle size and PDI value was selected. Results of stabilizer screening for the formulation of \u003cem\u003eC. limon\u003c/em\u003e nano suspensions are given in Table 1.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 1\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eScreening of stabilizers for the formation of \u003cem\u003eC. limon\u003c/em\u003e nanosuspension (Porwal, 2022)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eNanosuspension Code\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eStabilizer\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003ePhysical stability\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eZ-average size\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003ePolydispersity index\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eStability after 3 month (room temperature\u003c/p\u003e\n \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePVA\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eStable\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e129\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eUnstable\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eTween 80\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eStable\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e147\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eStable\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePEG\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eUnstable\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e580\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eUnstable\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eSLS\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eUnstable\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"char\"\u003e\n \u003cp\u003e557\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eUnstable\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eIt was apparent from the result that all formulated \u003cem\u003eC. limon\u003c/em\u003e nano suspensions with HPMC, PVA, PVP, tween-80 and PEG stabilizers were found physically stable when freshly prepared. But nano suspensions with HPMC and PVP stabilizers lost stability after one week (figure S1 in supplementary file). Therefore, only four nano suspensions were characterized. After characterization, nano suspension with PEG, SLS, PVA stabilizers were also lost stability after 3 month at room temperature (25\u003csup\u003eo\u003c/sup\u003eC). While nano suspension formulated with Tween-80 stabilizer remained stable even after 3 months. But Tween 80 stabilizer was selected for further optimization due to greater stability rate (Fig.S.6 in supplementary file). Figure 1 below shows particle size and PDI value of \u003cem\u003eC. limon\u003c/em\u003e nano suspension with Tween 80 stabilizer\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.2 Optimization of parameter\u003c/strong\u003es \u003cstrong\u003eof\u003c/strong\u003e \u003cstrong\u003eC. limon\u003c/strong\u003e \u003cstrong\u003enano suspension by response surface methodology (RSM)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eIn order to formulate therapeutically stable and efficient nanosuspension with the desired morphology and particle size, optimization of formulation parameters is required. After selecting a suitable stabilizer, various concentrations of the plant extract and stabilizer were optimized to achieve a nanosuspension with minimal particle size. The nano suspensions were formulated using a modified nano precipitation method (bottom-up approach). The formulation parameters, including the amount of plant extract (A), stabilizer concentration (B), and the S/AS ratio (C), were optimized through the central composite design of RSM. Optimization was focused on achieving the smallest particle size and lowest PDI value. Based on the response values obtained, the software predicted a quadratic model for the nanosuspension particle size. The influence of the independent variables on the response values were assessed by developing a regression equation for the response (Table S.2 and S.3 in supplementary file).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCitrus limon\u003c/strong\u003e \u003cstrong\u003eparticle size (nm) (R1)\u003c/strong\u003e = + 672.46 + 67.66A + 77.28B + 11.39C + 36.59AB + 79.56AC − 122.29BC − 109.91A\u003csup\u003e2\u003c/sup\u003e \u003cstrong\u003e− 1\u003c/strong\u003e53.41 \u003cstrong\u003e− 2\u003c/strong\u003e7.10C\u003csup\u003e2\u003c/sup\u003e \u003cstrong\u003e(1)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCitrus limon\u003c/strong\u003e \u003cstrong\u003ePDI (R2)\u003c/strong\u003e = + 0.1704–0.0854A + 0.0176B -0.0503C -0.1862AB -0.0617AC + 0.0383BC + 0.1684A\u003csup\u003e2\u003c/sup\u003e + 0.1394B\u003csup\u003e2\u003c/sup\u003e + 1861C\u003csup\u003e2\u003c/sup\u003e \u003cstrong\u003e(2)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe positive coefficients indicate a combined effect of the independent variables on reduction of particle size and PDI, while the negative values denotes an inverse effect. From the equations, it is evident that the amount of plant extract, concentration of stabilizer, solvent to anti-solvent ratio, and their interactions play crucial roles in decreasing the particle size and polydispersity index (PDI) of the \u003cem\u003eC. limon\u003c/em\u003e nanosuspension (Table .\u003c/p\u003e\n \u003cp\u003eANOVA was utilized to assess the linear, quadratic, and interactive effects of the independent variables on R1 and R2. The p-values (p = 0.0001) and F-values (61.93 for R1 and 88.20 for R2) demonstrated the significance of the quadratic model for optimizing the nanosuspension synthesis. P-values were used to determine the model's significance, with a p-value of \u0026lt; 0.05 indicating a significant model. The variables A and B, along with the interactions AC, BC, and the quadratic terms A² and B², had a significant impact on reducing the particle size of the \u003cem\u003eC. limon\u003c/em\u003e nanosuspension. Additionally, the parameters A, C, AB, AC, BC, A², B², and C² significantly affected the reduction in PDI, while the remaining parameters showed an inverse relationship with particle size and PDI (Tables 2 and 3).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eANOVA for quadratic model of particle size (R1) of \u003cem\u003eC. limon\u003c/em\u003e nano suspension (Uchida \u003cem\u003eet al.\u003c/em\u003e, 2021)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eSum of Squares\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003edf\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eMean Square\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eF-value\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e7.967E + 05\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e88523.94\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e61.93\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eA-Amount of plant extract\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e62526.86\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e62526.86\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e43.74\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eB-Amount of Stabilizer\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e81558.48\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e81558.48\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eC-Solvent to anti-solvent ratio\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1771.48\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1771.48\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1.2457.5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2917\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eAB\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10710.62\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10710.62\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e7.49\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0209\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eAC\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e50644.71\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e50644.71\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e35.43\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eBC\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1.196E + 05\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1.196E + 05\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e83.69\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eA²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1.741E + 05\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1.741E + 05\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e121.78\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eB²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e3.393E + 05\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e3.393E + 05\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e237.32\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eC²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10587.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10587.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e7.41\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0215\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eResidual\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e14295.21\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1429.52\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eLack of Fit\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e7025.07\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1405.01\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.9663\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5145\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003enot significant\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePure Error\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e7270.13\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1454.03\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eCor Total\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e8.110E + 05\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eR²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e0.9824\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eC.V. %\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e7.97\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePredicted R²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e0.9148\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eAdeq.\u0026nbsp;Precision\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e21.0933\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eAdjusted R²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e0.9824\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe strong predictability or consistency of the model was suggested by the non-significant lack of fit F values of R1 (0.96) and R2 (0.54). The coefficient of determination (R\u003csup\u003e2\u003c/sup\u003e) was used to provide an additional evaluation of the quadratic model's quality. The R\u003csup\u003e2\u003c/sup\u003e values for PDI (0.9876) and particle size (0.9824) indicated 98% variability in both responses. Because R2 was nearer 1.000, the model was more reliable and anticipated a better response. Regression models with R\u003csup\u003e2\u003c/sup\u003e \u0026gt; 0.9000 have historically been considered to have extremely strong correlation (Wu \u003cem\u003eet al.\u003c/em\u003e, 2023). According to Tables 2 and 3, the CV for R1 and R2 was determined to be 7.97 and 8.43%, respectively, and was considered satisfactory (Table S.4 in supplementary file).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 3\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eANOVA for quadratic model of PDI (R2) of \u003cem\u003eC. limon\u003c/em\u003e nanosuspension (Hazafa \u003cem\u003eet al.\u003c/em\u003e, 2022)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eSource\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eSum of Squares\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eDf\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eMean Square\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003eF-value\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\n \u003cp\u003ep-value\u003c/p\u003e\n \u003c/th\u003e\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eModel\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1.45\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1615\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e88.20\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eSignificant\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eA-Amount of plant extract\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0995\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0995\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e54.35\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eB-Amount of Stabilizer\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0042\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0042\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e2.30\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.1604\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eC-Solvent to anti-solvent ratio\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0345\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0345\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e18.85\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0015\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eAB\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2775\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2775\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e151.54\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eAC\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0305\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0305\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e16.66\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0022\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eBC\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0117\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0117\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e6.39\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0300\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eA²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4088\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4088\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e223.24\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eB²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2802\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.2802\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e153.00\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eC²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4991\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.4991\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e272.56\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt; 0.0001\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eResidual\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0183\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0018\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eLack of Fit\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0065\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0013\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.5499\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.7362\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003enot significant\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePure Error\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0118\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0024\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eCor Total\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e1.47\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eR²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e0.9876\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eC.V. %\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e8.43\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePredicted R²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e0.9549\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eAdeq.\u0026nbsp;Precision\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e27.5240\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eAdjusted R²\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e0.9764\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe impact of all independent variables on different responses of the \u003cem\u003eC. limon\u003c/em\u003e nano suspension was examined by using three dimensional response surface plots. In every plot, combined effect of two variables was simultaneously evaluated, whereas a third one was kept as constant. These plots evaluated that all three formulation parameters have a significant effect on reduction in particle size and PDI of \u003cem\u003eC. limon\u003c/em\u003e nanosuspensions (Table S.5 in supplementary file).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.2.1 Impact of amount of plant extract and conc. of stabilizer on particle size of\u003c/strong\u003e \u003cstrong\u003eC. limon\u003c/strong\u003e \u003cstrong\u003enanosuspension\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe graph clearly showed that when the stabilizer concentration decreased from 1 to 0.25g, there was a significant decrease in particle size. However, by increased the plant extract from 0.25g to 1 g, a significant decrease in particle size was also observed. Particle size (96.88 nm) significantly decreased at optimized conditions (1g plant extract and 0.25g concentration of stabilizer) by keeping solvent to anti-solvent ratio constant as showed in Fig. 2. A gradual increase in particle size was also examined by increasing the concentration of stabilizer from 0.25g. The explanation for decreasing particle size by increasing the polymer stabilizer concentration was that, an excess accumulation of polymer would enhance particle size and prevent the absorption during precipitation between solvent and anti-solvent. In addition, osmotic pressure increases by increasing the concentration of polymer. This leads to increased colloidal particle attraction, leading to greater size (Zhou \u003cem\u003eet al.\u003c/em\u003e, 2023). These results were correlated with previous findings like particle size of nanosuspension was increased by increasing the amount of plant extract and concentration of stabilizer from optimized conditions (Hou \u003cem\u003eet al.\u003c/em\u003e, 2022).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.2.2 Impact of amount of plant extract and conc. of stabilizer on PDI of\u003c/strong\u003e \u003cstrong\u003eC. limon\u003c/strong\u003e \u003cstrong\u003enanosuspension\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eFrom the graph, it was observed that minimum PDI value was observed at 0.25 g stabilizer concentration and 1 g amount of plant by keeping solvent to anti-solvent ratio constant (Fig. 3). When we increased stabilizer concentration and amount of plant extract from optimized conditions (0.25 g conc. of stabilizer and 1 g plant extract), PDI value also increased. Results of the current study correlated with previous findings which showed that optimized concentration of stabilizer control PDI value and minimum PDI was observed under optimum concentration of stabilizer (Van Driessche \u003cem\u003eet al.\u003c/em\u003e, 2022).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.3 Biological activities of\u003c/strong\u003e \u003cstrong\u003eC. limon\u003c/strong\u003e \u003cstrong\u003enanosuspension\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe following in-vitro biological assays were performed to check positive effective role of nanosuspension against bacteria, fungi and pests of agricultural origin.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\"\u003e\n \u003ch2\u003e3.3.1 Pesticidal activity\u003c/h2\u003e\n \u003cp\u003eThe repellent and pesticidal activity of \u003cem\u003eC. limon\u003c/em\u003e nanosuspension was observed against three pests like \u003cem\u003eTribolium castaneum\u003c/em\u003e (red flour beetle), \u003cem\u003eOryzaephilus surinamensis\u003c/em\u003e (sawtoothed grain beetle) and \u003cem\u003eSitophilus zeamais\u003c/em\u003e (corn weevil) under lab conditions. All treatments as well as control was applied in form of three replications. The \u003cem\u003eC. limon\u003c/em\u003e nanosuspension, the coarse peels extract and Malathion (control) were applied in five different concentrations such as 50, 100, 150, 200 and 250mg/ml while distilled water was used as negative control. After 24, 48 and 72 hours of treatment applications, mortality data was examined for three days.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eSitophilus zeamais\u003c/em\u003e or corn weevil is one of the primary stored grain pests. It mainly feeds on corn and damage crops like corn, grain, and cotton. \u003cem\u003eOryzaephilus surinamensis\u003c/em\u003e is a common secondary pest of cereals and cereal products and mostly found on copra, spices, nuts and dried fruit. Their adults and larvae are able to enter small cracks, so they can often attack packaged food or nuts in shell. \u003cem\u003eTribolium castaneum\u003c/em\u003e causes large economic losses of stored wheat grain. The major strategy used to protect stored crops is to employed chemicals in the form of pesticides. But excessive use of chemical insecticides caused environmental pollution and makes insect-resistant to a number of insecticides (Rauf, 2024). In this context, a new strategy should be analyze to combat major drawbacks associated with conventional insecticides. Due to enhanced rate of dissolution and solubility, increased stability and improved efficiency, nano suspensions become more reactive and this is one of the reasons for which it used as pesticides (Rani \u003cem\u003eet al.\u003c/em\u003e, 2023).\u003c/p\u003e\n \u003cp\u003ePesticides typically function by inhibiting or disrupting the translation process and protein synthesis, often through binding to prokaryotic ribosomes, which prevents peptide transfer and prevent chain elongation (Kocyigit \u003cem\u003eet al.\u003c/em\u003e, 2023). Nano bio pesticides have some added advantages over chemical and bio pesticides because of their increased solubilization abilities, bioavailability, stability, slow release, and targeted delivery, leading to enhanced efficiency. Studies on nano-formulations used against \u003cem\u003eTribolium castaneum\u003c/em\u003e have shown muscular destruction, pigmentation changes, and alterations in epidermal thickness, necrosis in the cuticle, and cellular damage in both the endocuticle and exocuticle. Ingestion of nano-biopesticides during early larval stages has been linked to impaired crawling and climbing abilities in later larval and adult stages (Bihal \u003cem\u003eet al.\u003c/em\u003e, 2023). Due to their bioavailability in plant systems, nano-biopesticides are valuable for understanding the interactions and behaviors of various pests that infest crops (Bihal \u003cem\u003eet al.\u003c/em\u003e, 2023)\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.3.2. Effect of\u003c/strong\u003e \u003cstrong\u003eC. limon\u003c/strong\u003e \u003cstrong\u003enanosuspension against sawtoothed beetle (\u003c/strong\u003e\u003cstrong\u003eO. surinamensis\u003c/strong\u003e\u003cstrong\u003e) and red flour beetle (\u003c/strong\u003e\u003cstrong\u003eT. castaneum\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eEffect of \u003cem\u003eC. limon\u003c/em\u003e nano suspension examined against saw-toothed beetle, red flour beetle and corn weevil to check the mortality rate of all thee insects. Twenty homogeneous adults were released in each petri-plate. These adults were allowed to feed on treated diet and mortality data was observed. The five varying concentrations were employed such as 50, 100, 150, 200, 250 mg/ml against saw-toothed beetle and red flour beetle. Three varying concentrations were employed such as 150, 200, 250 mg/ml against \u003cem\u003eS. zeamais\u003c/em\u003e and mortality data was examined at 24, 48 and 72 hours of exposure time. Figures 4, 5 and 6 shows Pesticidal activity of \u003cem\u003eC. limon\u003c/em\u003e peels coarse extract, \u003cem\u003eC. limon\u003c/em\u003e nanosuspension and Malathion (control) against \u003cem\u003eT. castaneum\u003c/em\u003e respectively\u003c/p\u003e\n \u003cp\u003eThe mortality rate of \u003cem\u003eC. limon\u003c/em\u003e nanosuspension against \u003cem\u003eO. surinamensis\u003c/em\u003e and \u003cem\u003eT. castaneum\u003c/em\u003e at various treatment and exposure of times showed in Table 4 and 5. \u003cem\u003eC. limon\u003c/em\u003e nanosuspension possessed significantly (p \u0026lt; 0.05) higher pesticidal activity than coarse peel extract against \u003cem\u003eT. castaneum\u003c/em\u003e. The maximum mortality rate of 97% was observed after 72 hours of exposure time whereas minimum mortality of 62% was observed for nanosuspension after 24 hrs. of exposure time at 250 mg/ml concentration. Similarly, at 250 mg/ml, the highest mortality rate of 45% was recorded for the coarse peel extract after 72 hours, compared to the lowest rate of 29% observed after 24 hours. This indicates that prolonged exposure leads to a gradual increase in insect mortality. Previous research showed that D-limonene could interfere with the pests octopaminergic system and cause nervous system damage (Ibrahium \u003cem\u003eet al.\u003c/em\u003e, 2022). The results of current investigation correlate with earlier reports in literature such as \u003cem\u003eC. limon\u003c/em\u003e EO-based nano emulsions were most efficient against \u003cem\u003eT. castaneum\u003c/em\u003e larvae by exhibiting 93.3% mortality rate in 14 days post-exposure (Papanikolaou \u003cem\u003eet al.\u003c/em\u003e, 2022). Figures 7, 8 and 9 shows Pesticidal activity of \u003cem\u003eC. limon\u003c/em\u003e peels coarse extract, \u003cem\u003eC. limon\u003c/em\u003e nanosuspension and Malathion (control) against \u003cem\u003eO. surinamensis\u003c/em\u003e respectively. Figures 10, 11 and 12 shows Pesticidal activity of \u003cem\u003eC. limon\u003c/em\u003e peels coarse extract, \u003cem\u003eC. limon\u003c/em\u003e nanosuspension and Malathion (control) against \u003cem\u003eS. zeamais\u003c/em\u003e respectively.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eC. limon\u003c/em\u003e nanosuspension also possessed significantly (p \u0026lt; 0.05) higher pesticidal activity than coarse peel extract against \u003cem\u003eO. surinamensis.\u003c/em\u003e The maximum mortality of 100% was observed after 24 hours of exposure time at highest concentration 250 mg/ml, whereas minimum mortality of 14% was observed after 24 hours of exposure time at lowest conc. of 50 mg/ml. Similarly, maximum mortality of 26% was recorded for the coarse peel extract after 72 hours, compared to the lowest rate of 15% observed after 24 hours. These results were correlated with the previous findings which showed that the mortality rate of pests was also increased gradually by increasing concentrations of nanosuspension (Palermo \u003cem\u003eet al.\u003c/em\u003e, 2021). But Malathion (control) showed significantly (p \u0026lt; 0.05) higher mortality rate than both \u003cem\u003eC. limon\u003c/em\u003e nanosuspension and coarse peels extract against \u003cem\u003eO. surinamensis\u003c/em\u003e and \u003cem\u003eT. castaneum\u003c/em\u003e and \u003cem\u003eS. zeamais\u003c/em\u003e as showed in Fig. 6.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 4\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffect of different exposure time and concentrations of \u003cem\u003eC. limon\u003c/em\u003e nanosuspensions and peels extract on mortality of \u003cem\u003eT. castaneum\u003c/em\u003e (El-Gendy \u003cem\u003eet al.\u003c/em\u003e, 2024)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eExposure time (h)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eConc. mg/ml\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e% Average Mortality ± SE\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eNanosuspension\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePlant extract\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive control\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative control\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10.0\u003csup\u003eCt\u003c/sup\u003e ± 0.5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e6.66\u003csup\u003eAcaCt\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e13.33\u003csup\u003eCt\u003c/sup\u003e ± 0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e16.66\u003csup\u003eCt\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e13.33\u003csup\u003eCt\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e18.33\u003csup\u003eCt\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e38.33\u003csup\u003eCopqr\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e30.0\u003csup\u003eCrs\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e41.6\u003csup\u003eCnopq\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e58.33\u003csup\u003eCijk\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e35.0\u003csup\u003eCpqr\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e61.6\u003csup\u003eChijk\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e76.33\u003csup\u003eCef\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e56.66\u003csup\u003eCijk\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e78.3\u003csup\u003eCdef\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e31.66\u003csup\u003eBqrs\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e21.66\u003csup\u003eBst\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e33.3\u003csup\u003eBpqr\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e38.33\u003csup\u003eBopqr\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e28.33\u003csup\u003eBrst\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e43.3\u003csup\u003eBmnop\u003c/sup\u003e ± 0.5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e61.66\u003csup\u003eBhijk\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e41.66\u003csup\u003eBnopq\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e63.33\u003csup\u003eBghij\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e73.33\u003csup\u003eBefg\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e51.66\u003csup\u003eBklmn\u003c/sup\u003e ± 0.6\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e78.33\u003csup\u003eBdef\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e83.33\u003csup\u003eBcde\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e61.66\u003csup\u003eBhijk\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e88.33\u003csup\u003eBbcd\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e51.66\u003csup\u003eAklmn\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e36.33\u003csup\u003eAopqr\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e56.6\u003csup\u003eAjkl\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e71.66\u003csup\u003eAfgh\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e46.6\u003csup\u003eAlmno\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e75.0\u003csup\u003eAef\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e78.33\u003csup\u003eAdef\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e53.\u003csup\u003e33Ajklm\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e83.3\u003csup\u003eAcde\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e91.66\u003csup\u003eAabc\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e61.66\u003csup\u003eAhijk\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e93.3\u003csup\u003eAabc\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e25\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e98.66\u003csup\u003eAab\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e68.33\u003csup\u003eAfghi\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eAa\u003c/sup\u003e + 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAu\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe % average mortality is given as mean ± SD. Means following same uppercase letter in each column are not substantially different. Within every row, means followed by the same lowercase letter are not significantly different; Tukey HSD test, p ˂ 0.05.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 5\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffect of different exposure time and concentrations of \u003cem\u003eC. limon\u003c/em\u003e nanosuspensions and peels extract on mortality of \u003cem\u003eO. surinamensis\u003c/em\u003e (Jampílek \u003cem\u003eet al\u003c/em\u003e., 2020)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eExposure time (h)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eConc. mg/ml\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e% Average Mortality ± SE\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eNanosuspension\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePlant extract\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive control\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative control\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e23.33\u003csup\u003eCqr\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e13.33\u003csup\u003eCr\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e28.33\u003csup\u003eCopq\u003c/sup\u003e ± 0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e38.33\u003csup\u003eClmno\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e25.0\u003csup\u003eCpqr\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e43.3\u003csup\u003eCjklm\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e71.66\u003csup\u003eCdef\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e33.3\u003csup\u003eCmnopq\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e75.0\u003csup\u003eCcde\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eCa\u003c/sup\u003e ± 0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e46.66\u003csup\u003eCijkl\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eCa\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eCa\u003c/sup\u003e ± 0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e55.0\u003csup\u003eCghij\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eCa\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e33.33\u003csup\u003eBmnopq\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e25.0\u003csup\u003eBpqr\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e36.6\u003csup\u003eBlmnop\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e58.33\u003csup\u003eBghi\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e31.6\u003csup\u003eBmnopq\u003c/sup\u003e ± 0.6\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e60.0\u003csup\u003eBfgh\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e76.66\u003csup\u003eBbcd\u003c/sup\u003e ± 1.0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e41.66\u003csup\u003eBklmn\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e78.33\u003csup\u003eBbcd\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eBa\u003c/sup\u003e ± 0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e55.0\u003csup\u003eBghij\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eBa\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eBa\u003c/sup\u003e ± 0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e61.66\u003csup\u003eBfg\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eBa\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e51.66\u003csup\u003eAghijk\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e30.0\u003csup\u003eAnopq\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e53.3\u003csup\u003eAghijk\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e78.33\u003csup\u003eAbcd\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e41.66\u003csup\u003eAklmn\u003c/sup\u003e ± 0.8\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e80.0\u003csup\u003eAbcd\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e86.66\u003csup\u003eAbc\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e48.33\u003csup\u003eAhijkl\u003c/sup\u003e ± 0.8\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e88.33\u003csup\u003eAab\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eAa\u003c/sup\u003e ± 0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e63.33\u003csup\u003eAefg\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eAa\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eAa\u003c/sup\u003e ± 0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e71.66\u003csup\u003eAdef\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eAa\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAt\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe % average mortality is given as mean ± SD. Means following same uppercase letter in each column are not substantially different. Within every row, means followed by the same lowercase letter are not significantly different; Tukey HSD test, p˂ 0.05.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3.3.3. Effect of\u003c/strong\u003e \u003cstrong\u003eC. limon\u003c/strong\u003e \u003cstrong\u003enanosuspension on corn weevil (\u003c/strong\u003e\u003cstrong\u003eS. zeamais\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eThe study revealed that \u003cem\u003eC. limon\u003c/em\u003e nanosuspension was also much potent against insect \u003cem\u003eS. zeamais\u003c/em\u003e and possessed significantly (p \u0026lt; 0.05) higher pesticidal activity than coarse peel extract. The maximum mortality of 100% was observed after 24 hours at highest concentration 250mg/ml, whereas minimum mortality rate 25% was observed after 24 hours of exposure time at 150mg/ml concentration. Similarly, after 72 hours of treatment with coarse peel extract, the mortality rate reached 23%. After 24 hours, the mortality rate was 10%. This indicates that longer exposure leads to a gradual increase in \u003cem\u003eS. zeamais\u003c/em\u003e mortality. But Malathion (control) showed significantly (p \u0026lt; 0.05) higher mortality rate than both \u003cem\u003eCitrus limon\u003c/em\u003e nanosuspension and coarse peels extract against \u003cem\u003eS. zeamais\u003c/em\u003e. Table 6 shows Effect of different exposure time and concentrations of \u003cem\u003eC. limon\u003c/em\u003e nanosuspensions and peels extract on mortality of \u003cem\u003eS. zeamais\u003c/em\u003e\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 6\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eEffect of different exposure time and concentrations of \u003cem\u003eC. limon\u003c/em\u003e nanosuspensions and peels extract on mortality of \u003cem\u003eS. zeamais\u003c/em\u003e (Gadelhaq \u003cem\u003eet al.\u003c/em\u003e, 2023)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eExposure time (h)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eConc. mg/ml\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"4\"\u003e\n \u003cp\u003e% Average Mortality ± SE\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eNanosuspension\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePlant extract\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003ePositive control\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003eNegative control\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e41.66\u003csup\u003eCde\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e26.66\u003csup\u003eCj\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e43.33\u003csup\u003eCcde\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCk\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e78.33\u003csup\u003eCcde\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e48.33\u003csup\u003eCj\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e80.0\u003csup\u003eCcd\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCk\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eCcd\u003c/sup\u003e ± 0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e58.33\u003csup\u003eCj\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eCbcd\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eCk\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e71.66\u003csup\u003eBbcd\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e38.33\u003csup\u003eBi\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e73.33\u003csup\u003eBabcd\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBk\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e88.33\u003csup\u003eBab\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e61.66\u003csup\u003eBhi\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e90.0\u003csup\u003eBabc\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBk\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eBa\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e71.66\u003csup\u003eBghi\u003c/sup\u003e ± 0.5\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eBa\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eBk\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e78.33\u003csup\u003eAabc\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e53.33\u003csup\u003eAgh\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e80.0\u003csup\u003eAa\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAk\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e91.66\u003csup\u003eAa\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e70.0\u003csup\u003eAfg\u003c/sup\u003e ± 0.57\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e93.33\u003csup\u003eAa\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAk\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eAa\u003c/sup\u003e ± 0\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e78.33\u003csup\u003eAef\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003csup\u003eAa\u003c/sup\u003e ± 0.3\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eAk\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe % average mortality is given as mean ± SD. Means following same uppercase letter in each column are not substantially different. Within every row, means followed by the same lowercase letter are not significantly different; Tukey HSD test, p ˂ 0.05.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\"\u003e\n \u003ch2\u003e3.4 Antibacterial activity\u003c/h2\u003e\n \u003cp\u003eThe antibacterial activity of \u003cem\u003eC. limon\u003c/em\u003e peels extract and their optimized nanosuspensions were investigated using the well diffusion method. Two bacterial strains such as \u003cem\u003eClavibacter michiganesis\u003c/em\u003e (gram-positive) and \u003cem\u003ePsedomonas syringae\u003c/em\u003e (gram negative) were used for this purpose. For antibacterial activity, Cciprofloxacin was used as a control while distilled water was used as negative control .The results were recorded in the absence or presence of an inhibition zone around the well. Each treatment was replicated three times. After treatments application, zone of inhibition were observed after 24 hours of exposure time. The antimicrobial activity was performed using three varying concentrations such as 150, 200, 250 mg/ml against two strains of bacteria such as \u003cem\u003ePseudomonas syringe\u003c/em\u003e and \u003cem\u003eClavibacter michiganesis\u003c/em\u003e.\u003c/p\u003e\n \u003cp\u003eBacterial speck, caused by \u003cem\u003eP syringae\u003c/em\u003e pathovar tomato (Pst), is a significant threat to tomato crops, potentially leading to a 75% yield loss if infection occurs during the seedling stage. The extensive use of chemical bactericides to manage this disease has led to pathogen resistance, environmental contamination, and health risks (Wang \u003cem\u003eet al.\u003c/em\u003e, 2023). Therefore, \u003cem\u003eC. limon\u003c/em\u003e nanosuspension has demonstrated effective results against this strain in preventing plant disease. Additionally, \u003cem\u003eC. michiganensis\u003c/em\u003e, the pathogen responsible for bacterial ring rot in potatoes (\u003cem\u003eSolanum tuberosum\u003c/em\u003e), is a notorious plant pathogen that has severely impacted potato cultivation. The extract from \u003cem\u003eC. limon\u003c/em\u003e peels has shown promising efficacy against this bacterial strains (Osdaghi \u003cem\u003eet al\u003c/em\u003e., 2022).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 7\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eAnti-bacterial activity against \u003cem\u003eP. syringae\u003c/em\u003e and \u003cem\u003eC. michiganesis\u003c/em\u003e (Arredondo-Valdés \u003cem\u003eet al\u003c/em\u003e., 2020)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAntimicrobial activity\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eConcentration (µg/ml)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eZone of inhibition (mm)\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eP. syringae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eC. michiganesis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eC. limon\u003c/strong\u003e \u003cstrong\u003ePlant Extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e8.46\u003csup\u003eCg\u003c/sup\u003e ± 0.2\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e5.6\u003csup\u003eCi\u003c/sup\u003e ± 0.15\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10.4\u003csup\u003eCf\u003c/sup\u003e ± 0.1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e10.46\u003csup\u003eCh\u003c/sup\u003e ± 0.25\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e15.7\u003csup\u003eCe\u003c/sup\u003e ± 0.2\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e12.53\u003csup\u003eCg\u003c/sup\u003e ± 0.25\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eC. limon\u003c/strong\u003e \u003cstrong\u003eNanosuspension\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e18.4\u003csup\u003eBe\u003c/sup\u003e ± 0.2\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e15.23\u003csup\u003eBf\u003c/sup\u003e ± 0.20\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e20.5\u003csup\u003eBc\u003c/sup\u003e ± 0.2\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e18.13\u003csup\u003eBe\u003c/sup\u003e ± 0.15\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e22.13\u003csup\u003eBb\u003c/sup\u003e ± 0.15\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e20.2\u003csup\u003eBc\u003c/sup\u003e ± 0.2\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003ePositive Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e21.4\u003csup\u003eAd\u003c/sup\u003e ± 0.1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e17.26\u003csup\u003eAd\u003c/sup\u003e ± 0.15\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e24.8\u003csup\u003eAb\u003c/sup\u003e ± 0.1\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e20.33\u003csup\u003eAb\u003c/sup\u003e ± 0.15\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e26.7 ± 0.25\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e22.6\u003csup\u003eAa\u003c/sup\u003e ± 0.1\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eNegative Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDj\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDj\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDj\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe zone of inhibition is given as mean ± SD. Means following same uppercase letter in each column are not substantially different. Within each row, means followed by the same lowercase letter are not significantly different; Tukey HSD test, p ˂ 0.05.\u003c/p\u003e\n \u003cp\u003eThe results of antibacterial activity showed that \u003cem\u003eC. limon\u003c/em\u003e nanosuspension showed significantly (p \u0026lt; 0.05) higher antibacterial activity against \u003cem\u003ePseudomonas syringae\u003c/em\u003e and \u003cem\u003eClabibacter michiganesis\u003c/em\u003e than peels extract of same concentration (Tables 7 and 8). \u003cem\u003eC. limon\u003c/em\u003e nanosuspension possessed significantly (p \u0026lt; 0.05) higher inhibition zone 22.13 ± 0.15 than \u003cem\u003eC. limon\u003c/em\u003e coarse peels 15.7 ± 0.2 whereas positive control Ciprofloxacin possessed maximum zone of inhibition 26.7 ± 0.25 against \u003cem\u003ePseudomonas\u003c/em\u003e. Ciprofloxacin showed significantly (p \u0026lt; 0.05) higher antibacterial activity (26.7 ± 0.25) in comparison to all other treatment groups in case of both strains. In case of \u003cem\u003eC. michiganesis\u003c/em\u003e (gram positive strain), maximum zone of inhibition manifested by \u003cem\u003eC. limon\u003c/em\u003e nanosuspension was 20.2 ± 0.2 and \u003cem\u003eC. limon\u003c/em\u003e coarse peels extract had 12.53 ± 0.25 zone of inhibition, whereas Ciprofloxacin (control) possessed 22.6 ± 0.1 zone of inhibition. This was clearly suggested that even low dosage of plant based nanosuspension has worked great and much compatible with market based product. Ciprofloxacin showed significantly (p \u0026lt; 0.05) higher anti-bacterial activity against \u003cem\u003eP. syringae\u003c/em\u003e and \u003cem\u003eC. michiganesis\u003c/em\u003e in comparison to all other treatment groups in case of both strains. 250mg/ml concentration of nano suspension was found out more effective against pesticides than other concentrations of nano suspension. Figures 13, 14 and 15 shows Zone of inhibition for \u003cem\u003eC. limon\u003c/em\u003e peels extract, \u003cem\u003eC. limon\u003c/em\u003e nanaosuspension and Ciprofloxacin (control) against \u003cem\u003eP. syringae\u003c/em\u003e and \u003cem\u003eC. michiganesis\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eHigher antibacterial activity of nanosuspensions can be due to higher dissolution rate and improved diffusion of nanosuspension in cultural media during experimental growth of bacteria (Singh \u003cem\u003eet al.\u003c/em\u003e, 2024). The strong reactivity and distinctive interactions of nanosuspensions with biological systems are a result of their distinctive physicochemical features (ultra-small size, large surface to mass ratio) (Marques and Kumar, 2023). This was concluded that the nanosuspension of \u003cem\u003eC. limon\u003c/em\u003e had quite comparable results with commercial antibiotics like Ciprofloxacin.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\"\u003e\n \u003ch2\u003e3.5. Antifungal activity\u003c/h2\u003e\n \u003cp\u003eAntifungal activity of \u003cem\u003eC. limon\u003c/em\u003e peels extract and their optimized nanosuspension was evaluated against two fungal strains such as \u003cem\u003eFusarium oxysporum\u003c/em\u003e and \u003cem\u003eRhizopus stolonifer\u003c/em\u003e by well diffusion method. The antifungal activity of \u003cem\u003eC. limon\u003c/em\u003e extract, its optimized nanosuspension and standard control (Iitraconazole) were evaluated at three different concentration such as 150, 200, 250 mg/ml. Iitraconazole was used as positive control while distilled water was used as negative control. 4.2g/100ml of Potato Dextrose Agar (PDA) was added in petri dishes and inoculated with fungal strains. Appropriately wells were impregnated with 30µl samples of treatment.\u003c/p\u003e\n \u003cp\u003eThe antifungal activities of treatments were examined after 48 hours by measuring zone of inhibition. The results of antifungal activity of optimized nanosuspension and coarse peels extract against both strains showed in Table 8. \u003cem\u003eC. limon\u003c/em\u003e nanosuspension possessed significantly (p \u0026lt; 0.05) higher antifungal potential than peels extract and control againt \u003cem\u003eF. oxysporum\u003c/em\u003e and \u003cem\u003eR. stolonifer\u003c/em\u003e. The results showed that \u003cem\u003eC. limon\u003c/em\u003e nanosuspension possessed maximum inhibition zone (25.46 ± 0.40) against \u003cem\u003eF. oxysporum\u003c/em\u003e at 250mg/ml concentration and minimum zone of inhibition (19.46 ± 0.25) was observed at 50mg/ml concentration than \u003cem\u003eC. limon\u003c/em\u003e coarse peels 18.6 ± 0.25 whereas positive control Ciprofloxacin possessed maximum zone of inhibition 0.83 ± 0.25 against \u003cem\u003eF. oxysporum\u003c/em\u003e. In case of \u003cem\u003eR. stolonifer\u003c/em\u003e, maximum zone of inhibition manifested by \u003cem\u003eC. limon\u003c/em\u003e nanosuspension was 20.12 ± 0.1 and \u003cem\u003eC. limon\u003c/em\u003e coarse peels extract had 13.33 ± 0.1 zone of inhibition, whereas Ciprofloxacin (control) possessed 0.96 ± 0.25 zone of inhibition.\u003c/p\u003e\n \u003cp\u003eWhereas, Fluconazole and Voriconazole (eminent antimycotic drugs) were used as controls, but surprisingly, they didn’t show any invitro antimycotic activity. But it was later found from a literature survey that the fungal species have developed resistance against the antimycotic drugs that were being used as antifungal control (Gupta \u003cem\u003eet al.\u003c/em\u003e, 2024). So, Itraconazole was showed better results as an antifungal control. Itraconazole showed significantly (p \u0026lt; 0.05) lower antibacterial activity in comparison to all other treatments in case of both strains Zone of inhibition increased with increased in the concentrations of \u003cem\u003eC. limon\u003c/em\u003e nanosuspension. Our results were closed to previous findings which examined that \u003cem\u003eC. limon\u003c/em\u003e peels extract caused 76.67% zone of inhibition against \u003cem\u003eF. oxysporum\u003c/em\u003e at 500 mg/ml (Gautam and Khedkar, 2024).\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab8\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 8\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003eAnti-fungal activity against \u003cem\u003eF. oxysporum\u003c/em\u003e and \u003cem\u003eR. stolonifer\u003c/em\u003e (Badaracco \u003cem\u003eet al.\u003c/em\u003e, 2020)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eAntimicrobial activity\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eConcentration (µg/ml)\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eZone of inhibition (mm)\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eF. oxysporum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eR. stolonifer\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eC. limon\u003c/strong\u003e \u003cstrong\u003ePlant Extract\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e14.5\u003csup\u003eCh\u003c/sup\u003e ± 0.2\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e9.33\u003csup\u003eCe\u003c/sup\u003e ± 0.1\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e16.35\u003csup\u003eCg\u003c/sup\u003e ± 0.2\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e11.53\u003csup\u003eCd\u003c/sup\u003e ± 0.1\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e18.63\u003csup\u003eCf\u003c/sup\u003e ± 0.25\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e13.33\u003csup\u003eCc\u003c/sup\u003e ± 0.1\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eC. limon\u003c/strong\u003e \u003cstrong\u003eNanosuspension\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e19.46\u003csup\u003eBe\u003c/sup\u003e ± 0.25\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e16.15\u003csup\u003eBi\u003c/sup\u003e ± 0.1\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e21.46\u003csup\u003eBd\u003c/sup\u003e ± 0.30\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e18.42\u003csup\u003eBh\u003c/sup\u003e ± 0.1\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e25.46\u003csup\u003eBb\u003c/sup\u003e ± 0.40\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e20.12\u003csup\u003eBg\u003c/sup\u003e ± 0.1\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003ePositive Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.78\u003csup\u003eAa\u003c/sup\u003e ± 0.26\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.73\u003csup\u003eAa\u003c/sup\u003e ± 0.26\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.87\u003csup\u003eAa\u003c/sup\u003e ± 0.37\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.85\u003csup\u003eAa\u003c/sup\u003e ± 0.37\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.90\u003csup\u003eAa\u003c/sup\u003e ± 0.25\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.96\u003csup\u003eAa\u003c/sup\u003e ± 0.25\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003e\u003cstrong\u003eNegative Control\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDi\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDj\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e200\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDi\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDj\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e250\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDi\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd align=\"left\"\u003e\n \u003cp\u003e0.00\u003csup\u003eDj\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eThe zone of inhibition is given as mean ± SD. Means following same uppercase letter in each column are not substantially different. Within every row, means followed by the same lowercase letter are not significantly different; Tukey HSD test, p ˂ 0.05.\u003c/p\u003e\n \u003cp\u003eIt was concluded from results that \u003cem\u003eC. limon\u003c/em\u003e nanosuspension possessed greater zone of inhibition against \u003cem\u003eF. oxysporum\u003c/em\u003e and \u003cem\u003eR. stolonifer\u003c/em\u003e than coarse peels extract. 250mg/ml concentration of nanosuspension was found out more effective against pesticides than other concentrations of nanosuspension. The biological active constituent of \u003cem\u003eC. limon\u003c/em\u003e peels is responsible for antifungal activity (Hou \u003cem\u003eet al.\u003c/em\u003e, 2022). Hence, plant-based nanosuspensions can be used as antifungal agents more effectively than commercial drugs due to their equivalent outcomes, cost effectiveness, high solubility, and ease of degradability. So, Present research work is in accordance with the previous study where the ultimate aim of researcher was to develop plant based formulation for treating plant disease and safe storage of grains (Albahri \u003cem\u003eet al.\u003c/em\u003e, 2023).\u003c/p\u003e\n \u003cp\u003eOur result were closed to previous study which studied that turmeric essential oil showed antifungal activity against mycelial growth and spore germination of \u003cem\u003eA. flavus\u003c/em\u003e in vitro (DEMIS and YENEWA, 2022).\u003c/p\u003e\n\u003c/div\u003e\n"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, the conventional use of chemical pesticides has significantly improved agricultural production but has raised environmental concerns. Nano biopesticides presents a promising solution, combining efficient pest control with enhanced properties such as durability and biodegradability. This study successfully formulated a nanosuspension of \u003cem\u003eC. limon\u003c/em\u003e using the nanoprecipitation method, demonstrating its positive impact on the health and growth of different plants in pot trials. The nanosuspension exhibited strong pesticidal activity highlighting its potential as an environmentally friendly and effective alternative for sustainable plant protection. With its notable stability and biodegradability, \u003cem\u003eC. limon\u003c/em\u003e plant-based nanosuspension emerges as a preferable choice for future agricultural practice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare the following financial interests/personal relationships which may be considered as potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUniversity has allowed us to conduct research on insects and pests in limited resource for wellbeing of human as well as for environment. During this research, anti-bacterial and anti-fungal activities were performed by using \u003cem\u003eC. limon\u003c/em\u003e nanobiopesticides.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study has been funded by Endowment Fund Secretariat University of Agriculture, Faisalabad, Pakistan, under grant No. RD-028-18.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003eThe following details on the availability of data were provide: The supplementary Files provide the raw measurements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publications:\u0026nbsp;\u003c/strong\u003eAll authors agree to publish.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate:\u0026nbsp;\u003c/strong\u003eNot applicable for this section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declaration:\u0026nbsp;\u003c/strong\u003eBefore participating in the study, every participant gave their informed permission, and the research was carried out in compliance with ethical guidelines and authorized by the appropriate institutional review board.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eHereby, we extend our gratitude to A. Q Research Group, Pakistan for reviewing the article and providing helpful comments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u0026nbsp;\u003c/strong\u003eAll authors contributed to the study conception and design. Methodology, data duration, investigation, and software were performed by Rabia Bibi. Project administration, visualization, and supervision were performed Nazish Jahan. Conceptualization, methodology, investigation, and validation were performed by Rabia Bibi. Review and editing were performed by Kousar Rasheed, Samiah Samiah and Aqsa Hameed. The first draft of the manuscript was written by Rabia Bibi, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdollahdokht, D., Y. Gao, S. Faramarz, A. Poustforoosh, M. Abbasi, G. Asadikaram and M. H. Nematollahi. 2022. Conventional agrochemicals towards nano-biopesticides: an overview on recent advances. Chem. Biol. Technol. Agric. 9:1-13.\u003c/li\u003e\n\u003cli\u003eAgbangba, C. E., E. S. Aide, H. Honfo and R. G. Kakai. 2024. On the use of post-hoc tests in environmental and biological sciences: A critical review. Heliyon 1:20-40. \u003c/li\u003e\n\u003cli\u003eAisvarya, S., M. Kalyanasundaram, M. Kannan, P. Arunkumar, S. Preetha, K. Elango and K. Govindaraju. 2024. Comparative analysis of the insecticidal activity against Sitophilus oryzae (L.) and agro-morphological characteristics of maize using non-biogenic and biogenic ZnO nanoparticles. Environ. Sci. Nano, 11:2173-2187.\u003c/li\u003e\n\u003cli\u003eAkhavan-Mahdavi, S., R. Sadeghi, A. F. Esfanjani, S. Hedayati, R. Shaddel, C. Dima, N. Malekjani, S. Boostani and S. M. Jafari. 2022. Nanodelivery systems for d-limonene; techniques and applications. Food Chem. 384:132460-132479.\u003c/li\u003e\n\u003cli\u003eAlbahri, G., A. A. Alyamani, A. Badran, A. Hijazi, M. Nasser, M. Maresca and E. Baydoun. 2023. Enhancing essential grains yield for sustainable food security and bio-safe agriculture through latest innovative approaches. Agronomy 13:1709-1715.\u003c/li\u003e\n\u003cli\u003eAlmahdy, A. G., A. El-Sayed and M. Eltarahony. 2024. A novel functionalized CuTi hybrid nanocomposites: facile one-pot mycosynthesis, characterization, antimicrobial, antibiofilm, antifouling and wastewater disinfection performance. Microb. Cell Fact. 23:140-148.\u003c/li\u003e\n\u003cli\u003eAnbazhagan, S. 2024. Biophysical characterization of Brucella abortus S19 perand evaluation of cross protective efficacy in mice model. Indian Veterinary Research Institute.\u003c/li\u003e\n\u003cli\u003eArredondo-Vald\u0026eacute;s, R., J. C. Chac\u0026oacute;n-Hern\u0026aacute;ndez, F. Reyes-Zepeda, F. D. Hern\u0026aacute;ndez-Castillo, J. C. Anguiano-Cabello, R. T. Heinz-Castro and S. G. Mora-Ravelo. 2020. In vitro antibacterial activity of Magnolia tamaulipana against tomato phytopathogenic bacteria. Plant Prot. Sci. 56:4-10\u003c/li\u003e\n\u003cli\u003eAssadpour, E., A. Can Kara\u0026ccedil;a, M. Fasamanesh, S. A. Mahdavi, M. Shariat-Alavi, J. Feng, M. S. Kharazmi, A. Rehman and S. M. Jafari. 2024. Application of essential oils as natural biopesticides; recent advances. Crit. Rev. Food Sci. Nutrit. 64:6477-6497.\u003c/li\u003e\n\u003cli\u003eElmadawy, A. A., D. M. El-Talpanty, M. El-Ebiary, T. Ismail and A. F. Omar. 2024. Toxicity and morphological changes induced by diatomaceous earth \u0026amp; malathion against Tribolium castaneum. New Valley J. Agric. Sci. 4:1-12.\u003c/li\u003e\n\u003cli\u003eBabarinde, S. A., K. A. Kemabonta, O. Z. Olatunde, E. O. Ojutiku and A. K. Adeniyi. 2021. Composition and toxicity of rough lemon (Citrus jambhiri Lush.) rind essential oil against red flour beetle. Acta Ecol. Sin. 41: 325-331.\u003c/li\u003e\n\u003cli\u003eBadaracco, P., M. Sortino and R. N. Pioli. 2020. Study of plant origin compounds with potential antifungal action against pathogens of cultivated plants.\u003c/li\u003e\n\u003cli\u003eBashir, L., A. Abdulkadir, M. Shah, A. Hamisu, U. Sharif and A. Kamalu. 2020. Phytochemical screening and antifungal potentials of Citrus limon peels against Fusarium oxysporum and Rhizopus stolonifer causing rots in water melon (\u003cem\u003eCitrullus lanatus\u003c/em\u003e L.). J. Exp. Sci. 11:1-5.\u003c/li\u003e\n\u003cli\u003eBihal, R., J. M. Al-Khayri, A. N. Banu, N. Kudesia, F. K. Ahmed, R. Sarkar, A. Arora and K. A. Abd-Elsalam. 2023. Entomopathogenic fungi: an eco-friendly synthesis of sustainable nanoparticles and their nanopesticide properties. Microorganisms 11:1617-1625.\u003c/li\u003e\n\u003cli\u003eChaubey, M. K. 2022. Chapter-3 Biology of Common Stored-Grain Insect Pests. Lat. Trends Zool. Entomol. Sci: 31.\u003c/li\u003e\n\u003cli\u003eCun, D., C. Zhang, H. Bera and M. Yang. 2021. Particle engineering principles and technologies for pharmaceutical biologics. Adv. Drug Deliv. Rev. 174:140-167.\u003c/li\u003e\n\u003cli\u003eEl-Gendy, R. M., E. M. Abd-ElAzeem and S. N. El-Shafiey. 2024. Moringa oleifera and Ruta angustifolia fixed oils and a prospective method to obstruct pupal development in cotton leafworm. J. Saudi Soc. Agric. Sci. 23:184-191.\u003c/li\u003e\n\u003cli\u003eFerreyra-Suarez, D., O. Garc\u0026iacute;a-Depraect and R. Castro-Mu\u0026ntilde;oz. 2024. A review on fungal-based biopesticides and biofertilizers production. Ecotoxicol. Environ. Saf. 283:116940-116945.\u003c/li\u003e\n\u003cli\u003eGadelhaq, S. M., S. M. Aboelhadid, A.-A. S. Abdel-Baki, K. M. Hassan, W. M. Arafa, S. M. Ibrahium, S. Al-Quraishy, A. O. Hassan and S. G. Abd El-Kareem. 2023. Safety and Efficacy of Pure and a Nanosuspension of D-limonene for Controlling Pigeon Lice. J. Med. Entomol. 60:148-158.\u003c/li\u003e\n\u003cli\u003eGupta, H., P. Bhatnagar and L. Shahani. 2024. Teratogenic effects of voriconazole (anti-fungal drug) on Swiss albino mice. Reprod. Toxicol. 129:108681-108686.\u003c/li\u003e\n\u003cli\u003eHasaballah, A., T. Selim, M. Tanani and E. Nasr. 2021. Lethality and vitality efficiency of different extracts of Salix safsaf leaves against the house fly, Musca domestica L.(Diptera: Muscidae). Afr. Entomol. 29:479-490.\u003c/li\u003e\n\u003cli\u003eHazafa, A., N. Jahan, M. A. Zia, K.-U. Rahman, M. Sagheer and M. Naeem. 2022. Evaluation and optimization of nanosuspensions of Chrysanthemum coronarium and Azadirachta indica using Response Surface Methodology for pest management. Chemosphere 292:133400-133411.\u003c/li\u003e\n\u003cli\u003eHou, T., S. S. Sana, H. Li, Y. Xing, A. Nanda, V. R. Netala and Z. Zhang. 2022. Essential oils and its antibacterial, antifungal and anti-oxidant activity applications: A review. Food Biosci. 47:101712-101716.\u003c/li\u003e\n\u003cli\u003eHussein, N. N., K. Al-Azawi, G. M. Sulaiman, S. Albukhaty, R. M. Al-Majeed, M. Jabir, A. G. Al-Dulimi, H. A. Mohammed, N. Akhtar and R. Alawaji. 2023. Silver-cored Ziziphus spina-christi extract-loaded antimicrobial nanosuspension: overcoming multidrug resistance. Nanomed. 18:1839-1854.\u003c/li\u003e\n\u003cli\u003eIbrahium, S. M., A. A. Wahba, A. A. Farghali, A.-A. S. Abdel-Baki, S. A. Mohamed, S. Al-Quraishy, A. O. Hassan and S. M. Aboelhadid. 2022. Acaricidal activity of tea tree and lemon oil nanoemulsions against Rhipicephalus annulatus. Pathogens 11:1506-1515.\u003c/li\u003e\n\u003cli\u003eIqbal, H., N. Jahan, S. Ali, A. Shahzad and R. Iqbal. 2024. Formulation of Moringa oleifera nanobiopesticides and their evaluation against Tribolium castaneum and Rhyzopertha dominica. J. Plant Dis. Protect. 131:133-142.\u003c/li\u003e\n\u003cli\u003eJamp\u0026iacute;lek, J., K. Kr\u0026aacute;ľov\u0026aacute; and P. Fedor. 2020. Bioactivity of nanoformulated synthetic and natural insecticides and their impact on environment. Nanopesticides: From Research and Development to Mechanisms of Action and Sustainable Use in Agriculture. Springer, New York City. pp.165-225.\u003c/li\u003e\n\u003cli\u003eKarabulut, F., A. Khan, E. F. Abd_Allah, J. A. Parray and A. Hashem. 2024. Omics and other biotechnological tools for biopesticide and microbial bioremediation. In: Microbiome-Assisted Bioremediation. Elsevier, Netherlands. pp.103-128.\u003c/li\u003e\n\u003cli\u003eKocyigit, E., B. Kocaadam-Bozkurt, O. Bozkurt, D. Ağag\u0026uuml;nd\u0026uuml;z and R. Capasso. 2023. Plant toxic proteins: their biological activities, mechanism of action and removal strategies. Toxins 15:356-360.\u003c/li\u003e\n\u003cli\u003eLaoudi, T., K. Khelfane-Goucem, S. Hamani-Aoudjit, F. Chebrouk, T. Amrouche, L. Saher and A. Kellouche. 2023. Chemical composition of essential oils from the leaves of Schinus molle and Cupressus sempervirens and their insecticidal activity against Oryzaephilus surinamensis (Coleoptera: Silvanidae). J. Essent. Oil Bear. Plants 26:309-322.\u003c/li\u003e\n\u003cli\u003eLiu, C., J. Wang, W. Su, G. Chen and J. Zhang. 2024. Effects of mango and partridge tea extracts on microbial, physical, and chemical properties of tilapia fillets treated with in-package cold plasma during refrigerator storage. Food Qual. Saf. 8:fyae001-fyae006.\u003c/li\u003e\n\u003cli\u003eMagi, M. S., L. Lopez-Vidal, M. C. Garc\u0026iacute;a, C. C. Stempin, C. Marin, B. Maletto, S. D. Palma, J. P. Real and A. F. Jimenez-Kairuz. 2024. Organic solvent-free benznidazole nanosuspension as an approach to a novel pediatric formulation for Chagas disease. Ther. Deliv. 1:1-18.\u003c/li\u003e\n\u003cli\u003eMarques, S. M. and L. Kumar. 2023. Factors affecting the preparation of nanocrystals: characterization, surface modifications and toxicity aspects. Expert Opin. Drug Deliv. 20:871-894.\u003c/li\u003e\n\u003cli\u003eMiedaner, T. and M. M. Garbelotto. 2024. Human-mediated migration of plants, their pathogens and parasites. J. Plant Pathol. 1:1-25.\u003c/li\u003e\n\u003cli\u003eMorteza-Semnani, K., M. Saeedi, J. Akbari, M. Moazeni, H. Seraj, E. Daftarifard, M. Tajbakhsh, S. M. H. Hashemi and A. Babaei. 2021. Fluconazole nanosuspension enhances in vitro antifungal activity against resistant strains of Candida albicans. Pharm. Sci. 28:112-129.\u003c/li\u003e\n\u003cli\u003eOsdaghi, E., J. M. van der Wolf, H. Abachi, X. Li, S. H. De Boer and C. A. Ishimaru. 2022. Bacterial ring rot of potato caused by Clavibacter sepedonicus: a successful example of defeating the enemy under international regulations. Mol. Plant Pathol. 23:911-932.\u003c/li\u003e\n\u003cli\u003ePalermo, D., G. Giunti, F. Laudani, V. Palmeri and O. Campolo. 2021. Essential oil-based nano-biopesticides: Formulation and bioactivity against the confused flour beetle Tribolium confusum. Sustainability 13:9738-9746.\u003c/li\u003e\n\u003cli\u003ePapanikolaou, N. E., N. G. Kavallieratos, V. Iliopoulos, E. Evergetis, A. Skourti, E. P. Nika and S. A. Haroutounian. 2022. Essential oil coating: Mediterranean culinary plants as grain protectants against larvae and adults of Tribolium castaneum and Trogoderma granarium. Insects 13:160-165.\u003c/li\u003e\n\u003cli\u003ePark, J.-S., J.-H. Choi, M.-Y. Joung, I.-G. Yang, Y.-S. Choi, M.-J. Kang and M.-J. Ho. 2024. Design of High-Payload Ascorbyl Palmitate Nanosuspensions for Enhanced Skin Delivery. Pharmaceutics 16:171-176.\u003c/li\u003e\n\u003cli\u003ePerween, N., S. Alshehri, T. Easwari, V. Verma, M. Faiyazuddin, A. Alanazi and F. Shakeel. 2021. Investigating the feasibility of mefenamic acid nanosuspension for pediatric delivery: Preparation, characterization, and role of excipients. Processes 9:568-574.\u003c/li\u003e\n\u003cli\u003ePorwal, O. 2022. Box-Behnken Design-based formulation optimization and characterization of spray dried rutin loaded nanosuspension: State of the art. S. Afr. J. Bot. 149:807-815.\u003c/li\u003e\n\u003cli\u003eRani, N., A. Duhan, A. Pal, P. Kumari, R. K. Beniwal, D. Verma, A. Goyat and R. Singh. 2023. Are nano-pesticides really meant for cleaner production? An overview on recent developments, benefits, environmental hazards and future prospectives. Journal of Cleaner Production, 411: 137232.\u003c/li\u003e\n\u003cli\u003eRauf, S. 2024. Insecticide resistance in agricultural pests: challenges and solutions. Trends Biotech. Plant Sci. 2:36-41.\u003c/li\u003e\n\u003cli\u003eRaveau, R., J. Fontaine and A. Loun\u0026egrave;s-Hadj Sahraoui. 2020. Essential oils as potential alternative biocontrol products against plant pathogens and weeds: A review. Foods 9:345-365.\u003c/li\u003e\n\u003cli\u003eRennick, B. 2023. Truffles in Michigan: Impacts of Herbicides on Their Growth, Effectiveness of In-Field Inoculations, and the Discovery of a Local Truffle (Tuber rugosum). Michigan State University, USA. pp.15-20.\u003c/li\u003e\n\u003cli\u003eS\u0026aacute;nchez-Hern\u0026aacute;ndez, E., V. Gonz\u0026aacute;lez-Garc\u0026iacute;a, A. Palacio-Bielsa, B. Lorenzo-Vidal, L. Buz\u0026oacute;n-Dur\u0026aacute;n, J. Mart\u0026iacute;n-Gil and P. Mart\u0026iacute;n-Ramos. 2023. Antibacterial activity of Ginkgo biloba extracts against Clavibacter michiganensis subsp. michiganensis, Pseudomonas spp., and Xanthomonas vesicatoria. Horticulturae 9:455-461.\u003c/li\u003e\n\u003cli\u003eSingh, S., S. Sharma and A. K. Dubey. 2024. Enhanced Antibacterial and Antibiofilm Activities of Actinobacterial Therapeutic Metabolites Mediated Nanosuspension. Part. Syst. Charact. 1:2300196-2300200..\u003c/li\u003e\n\u003cli\u003eSummer, M., H. M. Tahir, S. Ali, S. Nawaz, R. Abaidullah, S. Mumtaz, A. Ali and A. H. Gormani. 2024. Nanobiopesticides as an Alternative and Sustainable Solution to Tackle Pest Outbreaks. J. Kans. Entomol. Soc. 96:112-136.\u003c/li\u003e\n\u003cli\u003eVan Driessche, A. E., W. L. Ling, G. Schoehn and M. Sleutel. 2022. Nucleation of glucose isomerase protein crystals in a nonclassical disguise: The role of crystalline precursors. Proc. Natl Acad. Sci.119:e2108674110-e2108674119.\u003c/li\u003e\n\u003cli\u003eVisakh, N. U., B. Pathrose, M. Chellappan, M. Ranjith, P. Sindhu and D. Mathew. 2022. Chemical characterisation, insecticidal and antioxidant activities of essential oils from four Citrus spp. fruit peel waste. Food Biosci. 50:102158-102163.\u003c/li\u003e\n\u003cli\u003eVishnu, M., K. Malaisamy, R. Soundararajan, A. Suganthi, A. Subramanian, M. Senthilkumar, K. Rameash, K. Madesh and G. Kasivelu. 2024. Nano-bioformulations: emerging trends and potential applications in next generation crop protection. Environ. Sci. Nano. 1:75-95.\u003c/li\u003e\n\u003cli\u003eWang, W., Y. Weng, T. Luo, Q. Wang, G. Yang and Y. Jin. 2023. Antimicrobial and the resistances in the environment: ecological and health risks, influencing factors, and mitigation strategies. Toxics 11:185-190.\u003c/li\u003e\n\u003cli\u003eWu, S., Y. Sun, W. Jia, F. Wang, S. Lu and H. Zhao. 2023. Estimation of Above-Ground Carbon Storage and Light Saturation Value in Northeastern China\u0026rsquo;s Natural Forests Using Different Spatial Regression Models. Forests 14:1970-1986.\u003c/li\u003e\n\u003cli\u003eZaman, N., M. Rizwan, A. Iqbal, A. Rauf, Y. S. Al-Awthan and O. Bahattab. 2024. Phytochemistry and Medicinal Uses of the Family Rutaceae. In: Phytochemical and Pharmacological Investigation of the Family Rutaceae. Apple Academic Press, Canada. pp.51-69.\u003c/li\u003e\n\u003cli\u003eZhou, X., L. Lei, Y. Zeng, X. Lu, F. Liang, L. Zhang and G. Lin. 2023. High salinity effects on the depletion attraction in colloid-polymer mixtures. J. Colloid Interface Sci. 631:155-164.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nanosuspension, Citrus limon peels, nano biopesticides, Zeta sizer, Morality rate, Tween 80 stabilizer, Particle size, RSM","lastPublishedDoi":"10.21203/rs.3.rs-6714957/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6714957/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePost-harvest losses due to stored grains being compromised by insects, microorganisms, and environmental factors pose a significant challenge. These losses canbe both quantitative, affecting nutritional value, and qualitative, leading to commercial loss. Effective pest control measures, including monitoring and early detection of infestations, are essential to protect stored grains.In this study, \u003cem\u003eCitrus limon\u003c/em\u003e peels were selected for their biological activity and commercial viability and were processed into a nano-suspension. The acute toxicity and repellency of thesenano-formulations were tested against key stored product pests such as \u003cem\u003eOryzaephilus surinamensis\u003c/em\u003e, \u003cem\u003eTribolium confusum\u003c/em\u003e, and \u003cem\u003eSitophilus zeamais\u003c/em\u003e, alongside their antimicrobial activities. The developed nano-suspension exhibited optimal physical characteristics (particle size = 147.4 nm; PDI = 0.47). This formulation was repellent over time in area preference bioassays. Laboratory trials demonstrated that \u003cem\u003eC. limon\u003c/em\u003e nano-suspension achieved a 100% mortality rate against \u003cem\u003eO. surinamensis\u003c/em\u003e and \u003cem\u003eS. zeamais\u003c/em\u003e, and an 80% mortality rate against \u003cem\u003eT. confusum\u003c/em\u003e. Additionally, the \u003cem\u003eC. limon\u003c/em\u003e peels-based nano-biopesticides exhibited high antimicrobial activity against bacterial pathogens (\u003cem\u003ePseudomonas syringae\u003c/em\u003eand \u003cem\u003eClavibacter michiganensis\u003c/em\u003e) and fungal pathogens (\u003cem\u003eFusarium oxysporum\u003c/em\u003e and \u003cem\u003eRhizopus stolonifer\u003c/em\u003e). These results indicate that \u003cem\u003eC. limon\u003c/em\u003e peels-based nano-biopesticides, with their high dissolution rate, serve as effective antimicrobial agents and repellents, offering a promising alternative to chemical pesticides for controlling phytopathogenic species.\u003c/p\u003e","manuscriptTitle":"C. limon Peels Based Nano-Bio pesticides: Formulation and Bioactivity against the Stored Pests and Microbes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-09 15:38:33","doi":"10.21203/rs.3.rs-6714957/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bfc4e31f-4c79-4832-b2a3-768b528ed2be","owner":[],"postedDate":"July 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-22T23:32:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-09 15:38:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6714957","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6714957","identity":"rs-6714957","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}