Biological and chemical characterization of Origanum vulgare and Ocimum Basilicum essential oils and their derived nanoemulsions

Essential oils (EOs) are widely attractive components for pesticide, antimicrobial, and insecticide formulations because they are natural, renewable, biodegradable, environmentally non-persistent, and safe for non-target organisms and humans^1,2,3. Several Lamiaceae family members, for instance, sage, mint, oregano, basil, rosemary, and thyme, have become essential parts of the Mediterranean diet and traditional medicine due to their aromatic essential oils (EOs) and bioactive properties^4,5. Basil (Ocimum basilicum L.) and oregano (Origanum vulgare L.), belonging to the Lamiaceae family, are widely known for their therapeutic properties^6,7. The remarkable group of aromatic plants known as basil (Ocimum spp.) has become economically significant, mainly because of its essential oils, which are used in perfumery, cooking, and medicine⁸.

Naturally derived EOs from Ocimum gratissimum and Ocimum basilicum (2.5%) were combined with polylactic acid as a matrix and developed insecticidal polymer pouches⁹. The O. basilicum (OB) EOs revealed promising antibacterial, antioxidant, and potential larvicidal properties against mosquitoes. It is a viable substitute medication for treating human ailments and controlling mosquitoes as larvicidal control¹⁰. The extracted oil of OB exhibited significant anticancer, antimicrobial, and antioxidant properties, as well as anti-diabetic and anti-obesity effects, which provided a foundation for future research¹¹. O. basilium and M. piperita EOs would provide an additional approach for managing Phthorimaea absoluta, which are considered the most destructive pests of tomatoes, causing 100% yield loss in the absence of control measures¹². OB EOs and their aqueous extracts have antibacterial, antidiabetic, hepatoprotective, and anti-inflammatory properties¹³. ‎OB EOs and methyl cinnamate exhibited antibacterial activity, including bacteria resistant to β-lactam antibiotics. The findings suggest it may be a viable substitute for beta-lactam antibiotics¹⁴. O. bascilicum EOs and their nanoemulsion (Nano-E) exhibited potential larvicide activity LC₅₀ 81.07 µg/mL, and 65.19 µg/mL, respectively. So, basil nanoemulsions (Nano-Es) displayed strong larvicidal effects¹⁵.

Origanum vulgare L. subsp. hirtum, commonly referred to as Greek oregano, is a perennial plant that belongs to the Lamiaceae family. This herb is widely used in the food and pharmaceutical industries¹⁶. On the other hand, dried herbs are used in food processing, for example, meat products, alcoholic beverages, milk products, and snack foods. Origanum spp. is used in detergents, soaps, perfumes, flavorings, and cosmetics as a fragrance in addition to other pharmaceutical industries¹⁷. Through a variety of mechanisms of action, plant EOs, such as those in the Lamiaceae family, can be environmentally benign substitutes for larvicidal chemicals that cause acute larval toxicity and/or growth-inhibitory effects on mosquito developmental stages. Carvacrol and carvacrol-rich oregano EO are potent plant-based larvicides at doses lower than the acute lethal ones¹⁸. The chemical composition and biological activities of EOs from two Origanum vulgare (OV) genotypes belonging to the carvacrol (CAR) and thymol (THY) chemotypes grown in different cultivation environments. Only at doses below 0.02% were EOs from two Origanum spp. genotypes able to prevent epithelial monolayer sealing and inhibit the adhesion of specific pathogens, but they were unable to demonstrate any appreciable anti-inflammatory effects. These findings suggest their potential application as control agents against various foodborne diseases¹⁹. When tested, oils of OV against bacteria (Bacillus subtilis) susceptible as well as resistant bacterial strains) at different morphological stages demonstrated high activity. Because of this, they offer a cheap source of naturally occurring antibacterial compounds that may be applied to pathogenic organisms²⁰.

Unfortunately, some special properties limit the use of EOs, especially those not protected from external factors²¹. Therefore, EOs are an oily phase that easily undergoes oxidative damage, but it will make a greater propensity to shield themselves against oxidative damage when formulated in nanoemulsion systems, become more resistant to external factors, and be active for a longer time when nanoemulsion is formulated²². Nano-E‎ was more effective, physically stable, and a good soluble in water²³. Nano-E‎ formulations have size droplet dimensions related to the microemulsions reaching less than 200 nm or less than 100 nm in some cases²⁴. Enough interfacial corrugations result in the creation of the drops²⁵. The delivery method of Nano-E‎ formulation closes the gap between the active component content and bioaccessibility, which increases the bioactivity and efficiency²⁶. Nano-E‎s exhibits better antimicrobial activity than emulsions due to nano-sized droplets²⁷.

Agriculture pests led to high primary yield losses (26%) and even higher secondary yield losses (38%)²⁸. Chemical pesticides are widely used to control pests. However, several disadvantages of using synthetic pesticides have led to the search for and development of eco-friendly measures for use in pest management and reducing the harmful effects of synthetic pesticides. According to various reports, the essential oils of medicinal plants have broad-spectrum activities against a variety of pests. Nevertheless, they don’t provide a clear picture of how and when, as well as a suitable way to use these essential oils in pest management. Therefore, we assumed that each essential oil has a specific bioactive constituent and should be introduced in a suitable formulation in a pest management program after determining the effective concentration and the appropriate time against each of the targeted pests. This formulation will provide the greatest effectiveness, wider applicability for controlling pests, and acceptable environmental protection. Therefore, the current study will investigate the non-formulated essential oils of OB and OV and their nanoemulsions (Nano-Es) against some important pests after chemical and biological characterization to achieve sustainable crop protection.

Plant materials

The sweet basil (Ocimum basilicum L., var. basilicum, Family Lamiaceae) and Greek oregano (Origanum vulgare var. hirtum, Family Lamiaceae) were collected from the cultivated field in Al-Sharqia Governorate in 2022 and identified by plant taxonomists in the Desert Research Center, Egypt, according to Tâckholm²⁹. Weeds of bindweeds (Convolvulus arvensis L., Family Convolvulaceae), ryegrass (Lolium temulentum L., Family Poaceae), white goosefoot (Chenopodium album L., Family Chenopodiaceae), and dodder (Cuscuta planiflora, Family Convolvulaceae) were collected from T. alexandrinum (annual clover) fields at Marriott Research Station and identified by comparison with that of a known plant specimen at Desert Research Center after.

Extraction of essential oils

The air-dried aerial parts of O. basilicum (OB) and O. vulgare (OV) were ground before being subjected to extraction throughout the hydro-distillation process for 48 h using a Clevenger-type apparatus. The crude oils were dried over anhydrous sodium sulfate and kept in sealed vials at 4 °C for further examination.

Preparation and characterization of Nano-Es

The oil phase includes OB or OV essential oil (10 ml), co-surfactant (glycerol; 5 ml), and surfactant (Tween 80; 5 ml), was mixed under stirring (500–1000 rpm) to generate nanoemulsions (Nano-Es 10%) as the oil phase. The final volume was 100 ml after vortexing many times while adding deionized water (aqueous phase) during preparation^23,30. This combination was sonicated for 30 min in an ultrasonic bath (LC 60 H, Elma) with a thermometer (keep the sample ≤ 40 °C) during preparation³¹. The prepared Nano-Es droplet size was measured using Nano DLS/Particle Size Systems (NICOMP, N3000, Inc., Santa Barbara, Calif., USA). The zeta Sizer nano (Malvern, UK), model: (Nano ZS, ZEN 3600), was used to determine zeta potential range (−200:200 mV). Transmission electron microscopy (JEOL JEM 21OO, JAPAN) is used to reveal the internal structure with indexing for the diffraction pattern of OB and OV nano-emulsions³². Physical stability tests were implemented as described by Sinha et al³³. via centrifugation at 2000 rpm for 10, 20, and 30 min. and thermal stability at 4 ± 1 °C, 25 ± 1 °C, and 50 ± 1 °C for 30 days.

Herbicidal assays of O. basilicum and O. vulgare EOs and their Nano-Es

Laboratory bioassay

The essential oils of OB and OV were applied at concentrations of 0, 6.25, 12.5, 25, and 50 ‎µl ml-¹, while their formulated Nano-Es at 0, 5, 10, 15, and 20 ‎µl ml-¹ in Petri dishes containing the seeds on two-layer filter paper. After sterilization, the seeds of C. arvensis, L. temulentum, C. album, C. planiflora, and T. alexandrinum were put into Petri dishes (10 seeds/dish). These Petri dishes were rearranged in a completely randomized design for seven days at 25 ± 0.2 °C and 12 h of light. For every species, this test was run three times, with four replications. The seedling and germination parameters were measured, and then the % of inhibition was calculated.

Greenhouse experiment

In net greenhouses, in each pot containing 5 kg of sandy soil, 10 seeds of C. arvensis, L. temulentum, and C. album were sown until the stage of four leaves. The pots were watered two times each week with suitable amounts. A glass sprayer was used to apply a foliar spray of EO and Nano-Es solutions at concentrations of 0, 25, and 50 ‎µl ml^{– 1}, giving each pot 10 ml of solution. The treated pots were placed in a complete block design with four replications. Ten days after treatment, above-ground parts were cut, and fresh and dry weight, as well as its Ch A, Ch B, and carotenoids, were measured.

Antifungal activity of O. basilicum and O. vulgare EOs and their Nano-Es

The fungal mold test strain Fusarium oxysporum was isolated from infected (dry rot) potatoes (Solanum tuberosum L.) cultivated in the El-Frafra region at the New Valley governorate and then purified and identified according to micro- and macroscopic identification^34,35. The activity of EOs and their Nano-Es by testing 1 ml was investigated at 0, 25, 50, 100, 150, and 200 ‎µl ml-¹ concentrations in each Petri plate against F. oxysporum fungi. These treatments were applied and mixed before PDA (Potato Dextrose Agar) solidification. Then, 1 cm diameter discs (created by a punch sterilized cork borer) of the isolated F. oxysporum incubated culture strain were placed in the middle of the plates. After being incubated at 25 ± 2 °C for 7 days, the culture plates were inspected to identify and quantify the growth zones.

Antibacterial activity of O. basilicum and O. vulgare EO and their Nano-Es

Erwinia carotovora strain culture isolated from infected potatoes (blackleg and soft rot) cultivated in the El-Frafra region at New Valley-Governorate. The E. carotovora isolated bacterial strain was morphologically examined, biochemically and physiologically tested, and identified^36,37,38. The agar well diffusion technique was employed to test the antibacterial properties of OB and OV against Erwinia carotovora bacterial strain (isolated and identified). One milliliter of fresh bacterial culture suspension, after being adjusted to 0.5 McFarland Standard (1.5 × 10⁸ CFU/ml), was seeded in the nutrient agar (NA) plate before solidification. After that, wells were created aseptically using a cork borer with a 6 mm diameter. Subsequently, 100 µl of extract concentrations of 0, 25, 50, 100, 150, and 200 µl ml⁻¹ were added to each appropriate well. DMSO was utilized as the control treatment, and fosfomycin was used as a positive control. After 24 h, the treated plates in triplicate were incubated at 37 °C ± 2 for 24 h. Then, the zone of inhibition, including the well diameter, was measured.

Identification of Origanum vulgare and O. basilicum essential oil components

The essential oils of OB and OV were identified by GC-MS (Thermo Scientific Corp., USA) at the National Research Center of Egypt. The GC-MS system was equipped with a TG-WAX MS column (30 m x 0.25 mm i.d., 0.25 μm film thickness). Analyses were performed by carrier gas helium with a 1.0 ml/min flow rate and a split ratio of 1:10 using the following program: 40 °C for 1 min; rising at 4.0 °C/min to 160 °C and held for 6 min; rising at 6 °C/min to 210 °C and held for 1 min. Mass spectra were obtained by electron ionization at 70 eV, using a spectral range of m/z 40–450. Identified compounds were by Wiley spectral and NIST libraries based on authentic chemicals³⁹.

Data and statistical analysis

The reduction percentage of the measured parameters was calculated from the following equations: (R %) = C-T/C*100; after calculating [C = Control] [T = Treatment], the logistic fit equation in Sigma plot 12.5 was used to determine the effective dosage (ED₅₀ values). Each bioassay was performed in a complete randomized block design in four replicates. These data were statistically analyzed using IBM SPSS software version 21, an ANOVA to see whether there were significant differences (p < 0.05) between treatments and groups using the Duncan post hoc test.

Pre-emergence efficacy of Origanum vulgare and O. basilicum EOs and their Nano-Es against plant species germination and seedling growth

The assay revealed that Nano-Es of OB caused a superior decrease in C. arvensis, L. temulentum, C. album, C. planiflora, and T. alexandrinum compared to their EO based on their EC₅₀ values (Table 1). The highest activity of EO was detected in C. planiflora root length by EC₅₀ value of 8.88 ‎µl ml-¹. However, the lowest response was observed in L. temulentum by an EC₅₀ value of 22.14 ‎µl ml-¹. Similarly, the highest activity of OB Nano-E was observed in C. planiflora root length by EC₅₀ of 5.55 ‎µl ml-¹. However, the lowest activity was recorded in L. temulentum germination by EC_50, reaching 14.81 ‎µl ml-¹. Therefore, C. planiflora was the most sensitive plant, and root length was the most sensitive trait for OB EOs and Nano-Es. The highest significant interaction of treatments x conc. was (F = 2.63, p ≤ 0.00) C. arvensis, (F = 3.90, p ≤ 0.00) L. temulentum, (F = 11.48, p ≤ 0.00) C. album, (F = 22.06, p ≤ 0.03) C. planiflora, (F = 8.41, p ≤ 0.00) T. alexandrinum respectively. The highest significant interaction of conc. x traits in the tested plants was (F = 18.16, p ≤ 0.00) C. arvensis, (F = 129.16, p ≤ 0.00) L. temulentum, (F = 69.53, p ≤ 0.00) C. album, (F = 523.61, p ≤ 0.00) C. planiflora (F = 231.91, p ≤ 0.00) T. alexandrinum respectively. Thus, the most significant response to O. basilicum EOs and Nano-E was observed prominently in the parameters of C. planiflora species as recorded in Table (1).

As for OV, the highest activity of EO was detected in C. planiflora root length by EC₅₀ of 11.14 ‎µl ml-¹. However, the lowest EC₅₀ was observed in C. album by 18.73 ‎µl ml-¹. Similarly, the highest activity of Nano-E was achieved in C. planiflora root length by EC₅₀ of 6.02 ‎µl ml-¹. However, the lowest response was observed in L. temulentum germination by EC₅₀ of 14.81 ‎µl ml-¹. The interactions of EO and Nano-E (treatments x conc.) in the tested plants were significant in (F = 13.47, p ≤ 0.02) C. album, (F = 25.88, p ≤ 0.00) C. planiflora, (F = 9.87, p ≤ 0.00) T. alexandrinum, respectively. The highest significant interaction of EO and Nano-E (treatments x conc.) in the tested plants was (F = 121.13, p ≤ 0.00) C. arvensis, (F = 151.55, p ≤ 0.00) L. temulentum, (F = 81.58, p ≤ 0.00) C. album, (F = 614.38, p ≤ 0.00) C. planiflora, (F = 272.12, p ≤ 0.00) T. alexandrinum, respectively. Consequently, the most observed response to O. vulgare EOs and Nano-E was clearly significant in the characteristics of C. planiflora species, followed by T. alexandrinum and C. album, and finally by L. temulentum and C. arvensis as represented in Table (2).

Post-emergence activities of O. basilicum and O. vulgare EOs and their Nano-Es against weeds growth traits

The post-emergence activities of OB EO and Nano-E were evaluated on C. arvensis, L. temulentum, and C. album seedlings at the 4 leaf stage under greenhouse conditions (Table 3). As for EO activity, the highest concentration (50 ‎µl ml-¹) caused reduction in fresh weight, dry weight, Ch A, Ch B and carotenes by 81.72, 60.62, 55.94, 57.70 and 46.88% (C. arvensis), 70.41, 41.09, 55.54, 60.46, and 49.56% (L. temulentum), 58.01, 38.93, 54.41, 59.80, and 46.27% (C. album) respectively, over the control. As for Nano-E, the highest concentration resulted in inhibition in fresh weight, dry weight, Ch A, Ch B, and carotenoids by 82.95, 62.60, 123.17, 57.68 and 48.79% (C. arvensis), 77.54, 50.36, 54.89, 57.25 and 48.88% (L. temulentum), 64.10, 41.98, 54.98, 57.68, and 50.60% (C. album) respectively, relative to the control. The efficiency was affected significantly by the interactions of species x concentration for EO; (F = 626.90, p ≤ 0.00) fresh weight, (F = 23.21, p ≤ 0.00) fresh weight, (F = 62.66, p ≤ 0.00) Ch A, (F = 8.23, p ≤ 0.000) Ch B, and (F = 84.29, p ≤ 0.00) carotenoids, respectively. While for Nano-E was (F = 742.093, p ≤ 0.000), fresh weight (F = 6.233, p ≤ 0.00), dry weight (F = 8.62, p ≤ 0.000), Ch A (F = 7.97, p ≤ 0.00), and Ch B (F = 32.88, p ≤ 0.00) carotenoids, respectively. Accordingly, Nano-E of O. basilicum exhibited the most significant post-emergence activity among EOs in the tested species, especially in C. arvensis (Table 3).

The post-emergence activities of OV against C. arvensis, L. temulentum and C. album seedlings revealed that the highest concentration of EO caused a reduction in fresh weight, dry weight, Ch A, Ch B, and carotenes by 81.72, 60.63, 55.94, 59.63, and 46.88% (C. arvensis), 70.41, 41.09, 55.54, 60.46, and 49.56% (L. temulentum), 65.98, 38.93, 54.41, 59.80, and 46.27% (C. album) respectively inhibition relative to the control. As for Nano-E, the highest concentration resulted inhibition in fresh weight, dry weight, Ch A, Ch B and carotenoids by 82.95, 62.60, 55.19, 57.70, and 48.79% (C. arvensis), 77.54, 50.36, 54.89, 57.25, and 48.88% (L. temulentum), 64.10, 41.98, 54.98, 57.68, and 50.60% (C. album) relative to the control, respectively. The efficiency was affected significantly by the interactions of species x concentration; (F = 681.66, p ≤ 0.00) fresh weight, (F = 25.23, p ≤ 0.00) fresh weight, (F = 68.11, p ≤ 0.00) Ch A, (F = 8.96, p ≤ 0.000) Ch B, and (F = 91.62, p ≤ 0.00) carotenoids respectively for EO, while it was (F = 806.65, p ≤ 0.000) fresh weight, (F = 6.77, p ≤ 0.00) dry weight, (F = 9.37, p ≤ 0.000) Ch A, (F = 8.66, p ≤ 0.00) Ch B and (F = 35.75, p ≤ 0.00) carotenoids respectively. As a result, Nano-E of O. vulgare showed the most significant post-emergence activity than EOs in the tested species, especially in C. arvensis (Table 4).

The antifungal activity of O. basilicum and O. vulgare EOs and their Nano-Es against F. oxysporum fungi

The antifungal efficiency of OB and OV EOs and their Nano-Es were investigated in Table (5). Doses of 0, 25, 50, 100, 150, and 200 ‎µl ml-¹ were used to test the antifungal efficacy of EOs and their Nano-Es against F. oxysporum fungus. The EC₅₀ of O. basilicum EO and its Nano-E was achieved by 114.89 and 54.94 ‎µl ml-¹, respectively. While OV both EO and its Nano-E achieved EC₅₀ by 89.56 and 33.54 ‎µl ml-¹, respectively. These results demonstrate that by increasing the concentration of OB and OV EOs and their Nano-Es, the growth of F. oxysporum decreases and vice versa. As a result, O. vulgare showed more significant activity than O. basilicum in F. oxysporum (Table 5).

The antibacterial activity of EOs and their Nano-Es against E. carotovora

Using disc diffusion techniques, the antibacterial properties of OB and OV EOs and Nano-Es were evaluated against E. carotovora (Table 6). O. basilicum EO and its Nano-E achieved EC₅₀ by 179.76 and 128.65 ‎µl ml-¹, respectively. The efficiency of OV EO and their Nano-E presented from an inhibition zone that had EC₅₀ by 171.76 and 114.65 ‎µl ml-¹, respectively. This study has proven that the increase in concentration of EOs and Nano-Es of OB and OV directly correlates with increased activity against E. carotovora. The OV had higher antibacterial activity compared to OB, as indicated by EC₅₀ values, as shown in Table 6.

The composition of O. basilicum and O. vulgare essential oils by GC/MS

The constituent analysis identified 37 and 40 compounds in OB and OV EOs, respectively, which were quantified at 0.65% and 0.85% (v/w), respectively. The major compounds in OB were geranyl acetate (15.58%), linalool (13.68%), methyl chavicol (9.56%), terpinene-4-ol (9.10%), Beta-myrcene (7.40%), and 1, 8-cineol (6.76%), respectively. It was found that O. vulgare EO contained high amounts of carvacrol (13.565%), thymol (11.78%), dihydrocarveol (11.36%), carvacrol methyl ether (7.60%), α-terpinene 7.09% α- and cymene 6.61% and pinene (6.03%), respectively, as determined by GC/MS (Table 7). These compounds are related to different chemical groups, including monoterpene hydrocarbons (α-pinene, β-pinene, limonene), oxygenated monoterpenes (linalool, geraniol), sesquiterpene hydrocarbons (caryophyllene, humulene), oxygenated sesquiterpenes (nerol, bisabolene), diterpenes (terpinyl acetate), phenylpropanoids (cinnamaldehyde), aliphatic compounds (terpinyl acetate), and others (Table 7).

Nano-E characterization prepared from essential oils

The Nano-E of OB is characterized by nanoparticle size (13.45 ± 6.3 nm), Chi Sq = 14.45, at temperature (23 °C), and viscosity (0.933 cp.), respectively. The OV Nano-E was characterized by nanoparticle size (22.4 ± 8.0 nm), Chi Sq = 17.9, at temperature (23 °C) and viscosity (0.933 cp.), respectively. These Nano-Es have good physical stability, no aggregation, and separation parts under visual inspection to observe any changes in the Nano-E‎ stability under laboratory conditions Fig. 1. The transmission electron microscopy (TEM) images of OB and OV Nano-Es have an oil scale bar of 46.76 and 57.32 nm, with the interface between oil-water scale bars at 14.27 to 14.86 nm (Fig. 2). Consequently, the optimization impact of Nano-E was notably greater in OB compared to OV, as documented in figures (1& 2).

Nano-E‎s of O. basilicum and O. vulgare size by droplet size analyzer.

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Scales of the prepared Nano-Es by TEM scan.

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Zeta potential of O. basilicum and O. vulgare Nano-Es

The zeta potential of OB and OV Nano-Es is considered strongly anionic at approximately − 27.0 and 27.6 mV (Fig. 3). The prepared Nano-Es were subjected to the stability assessment of centrifugation at 2000 rpm for 10, 20, and 30 min. At the same time, thermal stability was implemented for 30 days. These Nano-E‎s have good physical stability toward precipitation after 10 and 20 min of centrifugation. However, trace precipitation in most prepared formulations at 30 min. The thermal stability studies of the tested Nano-Es were stable at 4 ± 2, 25 ± 2, and 40 ± 2 °C for thirty days. However, there was no aggregation and separation under visual inspection to observe any changes in the Nano-E‎ stability (Table 8). Accordingly, the developed Nano-E of OB and OV demonstrated evident physical stability under the parameters tested (Fig. 3; Table 8).

Zeta potential analysis of Nano-Es scales.

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The current study aims to use essential oils of OB and OV as sources of active substances to prepare natural formulations as alternative tools to synthetic pesticides in pest management. Essential oils (EOs) represent a complex range of secondary metabolites and interact synergistically to enhance their deleterious effects on insects⁴⁰. Eugenol, 1, 8-cineole, and linalool are derived from OB EOs constituents and have antimicrobial effects⁴¹. O. basilicum EO activity according to the most active common components, including terpineol, limonene, linalool, methyl cinnamate, eugenol, chavicol, and methyl eugenol⁴². OB presented opportunities for its usage as a source, mainly at the flowering stage, to extend new medicines based on natural active components⁴³. O. vulgare EO is rich in carvacrol, which has revealed antimicrobial actions against pathogenic microbes⁴⁴. Carvacrol is a compound found in the maximum quantity in OV EO and seems to indicate no major or marginal toxic effects in vivo⁴⁵. Carvacrol has antimicrobial activity; therefore, it deserves prominent scientific interest as a natural antimicrobial agent⁴⁶. However, EOs are one of the most highly volatile and susceptible to environmental degradation, including light, moisture, or air⁴⁷. Therefore, the essential oils were used to prepare nano-emulsions (Nan-Es), which have remarkable characteristics for agriculture applications. The efficacy of both prepared EOs and their Nano-Es were evaluated against several pests, including weeds of C. arvensis, L. temulentum, C. album, and F. oxysporium fungi, as well as E. carotovora bacteria, to determine the effective concentrations and application.

The multivariate analysis indicated that the tested weed species were affected significantly by the EOs and their Nano-Es in the pre-emergence and post-emergence tests. The activities of EO against C. arvensis, L. temulentum, C. album, C. planiflora, and T. alexandrinum depended on the concentration used. Whereas C. planiflora was the most susceptible plant to both OB and OV EOs and their Nano. However, C. arvensis and L. temulentum appeared to be less susceptible plants, respectively. In this issue, root length was the most sensitive trait compared with germination percentage and shoot length. In the post-emergence test, OB and OV Nano-Es have more herbicidal activities more than their essential oils against C. arvensis, L. temulentum, and C. album seedlings at the 4 leaf stage. C. album was more susceptible than C. arvensis, followed by L. temulentum weeds. Whereas OB was more effective than OV in both EOs and Nano-Es. In the pre- and post-emergence applications, the formulated Nano-Es have higher activity than non-formulated essential oils based on the weed response and the biochemical composition, respectively. The herbicidal efficacy of essential oils is derived from their interaction with cell membranes, inducing alterations in the lipid bilayer⁴⁸. Nanotools in weed management can minimize costs and environmental effects compared to the use of chemical herbicides⁴⁹.

The antifungal properties of OB and OV EOs and their Nano-Es were tested against wilt disease caused by F. oxysporium. The results showed that OV was more effective than OB, whereas the formulated Nano-Es has a higher effect than EOs on F. oxysporium. Nano-emulsions of essential oils are useful for controlling fungal growth because they can be used to cover, protect, and deliver a variety of natural substances⁵⁰. Carvacrol and thymol, usually the main volatiles in oregano EOs, have potent anthelmintic, fungicidal, and other bioactive properties⁵¹. The greatest antifungal activity of EOs for different basil varieties has been demonstrated⁵². OB is rich in EOs and phenolic compounds. These compounds are well known for their antifungal activity⁵³. Oregano essential oils have antibacterial and antifungal activities⁵⁴. OB crude extract, including EO, has important antifungal activity against fungi and other phytotoxic substances⁵⁵.

OB and OV EOs and their formulated Nano-Es have antibacterial activity towards E. carotovora isolated from potato tubers. The results displayed the higher efficacy of OV than OB. While Nano-E‎ had the highest activity compared to essential oils. Nano-E‎ droplets of oregano EO, ranging in size from 180 to 250 nm, integrated with edible methylcellulose films had improved antimicrobial efficacy. They prevented the growth of yeast and mold and improved the shelf life of sliced bread⁵⁶. O. vulgare EO produced inhibition zone diameters ranging from 24.6 to 34.1 mm, on average, and MBC ranging from 3.9% to 15.6% in Vibrio parahaemolyticus and Vibrio vulnificus, respectively⁵⁷. The oregano oil showed a powerful bacteriostatic and bactericidal effect⁵⁸. O. basilicum EO of this region contains methyl eugenol/methyl chavicol chemotype and has bactericidal properties⁵⁹. The antimicrobial activity of EO seems to depend on the presence of certain components that vary within the same species of plant⁶⁰. EO can directly impair the cell membrane of bacterial gram + ve, causing breakage of the bacterial cell membrane, blocking enzyme systems, and disrupting ion exchange⁶¹. Excellent anti-inflammatory and antifungal responses were shown by the majority of essential oils; moreover, T. vulgaris and O. vulgare also showed antibacterial activity⁶².

The studied EOs contain a high content of phenolic monoterpene compounds that are responsible for their herbicidal, antifungal, and antibacterial activity. These natural constituents are useful components within the prepared formulations of Nano-E‎s that have remarkable efficiency when applied to the treatment of weeds, fungi, and bacteria. Among the plant families demonstrating promise, EOs used as herbicides, including Myrtaceae, Lamiaceae, Asteraceae, and Anacardiaceae, are the compounds most often derived from mixtures with high activity, including limonene, α-pinene, 1, 8-cineole, camphor, carvacrol, and thymol⁶³. Compound 1,8-cineole inhibits seed germination, seedling growth, speed of germination, chlorophyll content, and respiratory activity of Ageratum conyzoides⁶⁴.

In this work, formulating EOs into nano-emulsions is very important to facilitate their application in pest control. In these nano-formulations, 10% essential oil was used with a tiny droplet of surfactant and co-surfactants in water after being exposed to ultra-sonication. The formulated Nano-E was characterized by its nanoparticle size, viscosity, TEM, zeta potentials, and physical stability. Therefore, Nano-Es were suitable in agricultural applications due to better stability than EOs for pest control. These formulations can enhance the activities, preserve the bioactive constituents, and broaden their usage for several applications. The formulated Nano-E‎s size was found close to the standard Nano-Es between 1 and 100 nm for the best delivery of their function⁶⁵. Despite their metastability, Nano-E‎s can persist over many months or years due to the presence of a stabilizing surfactant that inhibits the coalescence of the droplets⁶⁶. Nano-E‎s can be used as microreactors of controlled size for the preparation of monodisperse particles⁶⁷. To confirm the stability of the prepared Nano-E‎s of OB and O. vulgare for agricultural applications, the generated Nano emulsion droplet size was subjected to physical and chemical characterization. Droplet sizes, zeta potentials, polydispersity index, stability, viscosity, and morphologies of particles are the characteristic properties of Nano-E‎s⁶⁸. The tested nano-emulsions are more active than their EOs against the target pests. The Nano-Es toxicity is complex and based on different physicochemical characteristics, including size, shape, charge, and reactivity⁶⁹. Droplet size reduction improved the bioactivity of oregano essential oil, carvacrol, and thymol, suggesting that Nano-E‎-based delivery systems for natural compounds may be alternatives for food applications⁷⁰.

Nanoemulsion formulation is considered an efficient and versatile delivery method for certain molecules, serving as an effective carrier system. Its advantages are predominantly based on three key properties: a high surface-to-volume ratio, reduced droplet size, and enhanced stability. These emulsions are particularly well-suited for encapsulating lipophilic or hydrophobic essential oils that have low water solubility. The high surface-to-volume ratio of these minute oil droplets enhances the compound’s bioavailability and effectiveness. The creation of essential oil nanoemulsions improves their functional efficiency, scalability, and capacity to produce nano-sized droplets with a narrow size distribution. However, achieving emulsification necessitates a high surfactant and co-surfactant content to form stable nanoemulsions. In addition to stabilizing the essential oils, this process acts as a post-treatment that can enhance their physical, electrophoretic, interfacial, biochemical, and biological properties^{71,72,73,74,75}.

Due to their lipophilic nature, essential oils exert their antimicrobial action primarily by affecting the cellular membranes of target organisms⁷⁶. The nanoemulsion system an oil-in-water formulation enhances this mechanism of action. It stabilizes the lipophilic compounds, can impart hydrophilic activity, and encapsulates the bioactive compounds. This leads to increased membrane disruption and altered membrane permeability^77,78.

Finally, OB and OV essential oils and their preparation of Nan-E formulations reported remarkable activities against agricultural pests. The prepared Nano-E‎s were more biologically active as a natural pesticide against weeds of C. arvensis, L. temulentum, C. album, C. planiflora, and fungi (F. oxysporium), as well as bacteria E. carotovora. The most sensitive plant was C. planiflora; however, C. arvensis and L. temulentum revealed more tolerable weeds to OB and OV, respectively, without any selectivity on T. alexandrinum crop. Therefore, a selective inter-row application in crops or between rows of fruits is preferred to suppress weed species safely without damage. Both EOs and nano-emulsion activities depended on the target species, the application dose, and the application time, for which pre-emergence could be preferred over post-emergence application. Also, nano-emulsions are more active and could be helpful in applications of EOs. Consequently, the findings demonstrated that the potential use of prepared Nano-E‎s from essential oils of OB and OV was reliable as broad-spectrum natural pesticides and alternatives to some synthetic pesticides.

It could be concluded from the above results that, by comparing synthetic pesticides with nano-emulsions (Nano-Es), while initial production costs for Nano-Es may be greater, variables including consumer demand for natural products and environmental benefits may outweigh these costs. Overall, synthetic pesticides are more cost-effective and effective. With proven effectiveness and enhanced safety profiles, it is anticipated that EO-based Nano-Es will offer a viable, environmentally benign substitute for synthetic pesticides as long-term pest management options. Their incorporation into pest management plans may be justified by the advantages they provide for the environment and human health. The consistency in essential oils and their nano-emulsions is crucial for reliable pest control due to oil variability, nano-emulsion stability, and implementing good agricultural practices and their effectiveness in pest management. For scaling up Nano-Es production, these require meticulous planning to ensure consistency, efficiency, and cost-effectiveness. Ultrasonication could commonly be employed to facilitate the creation of stable Nano-Es with uniform droplet sizes, essential for efficacy and desired characteristics, such as droplet size and stability. Also, we should develop strong experimental protocols to assess the efficacy of Nano-Es against target pests under field experiments. By following this roadmap, successful Nano-Es will transition to market-ready products as alternatives to synthetic pesticides. On the other hand, evaluating the risks associated with the use of nanoemulsions (Nano-Es) is very important. Collaboration between researchers, industry stakeholders, and regulatory bodies is crucial to ensure the safe and sustainable use of nanoemulsions. ‎This research opened the way for specific future research directions such as (i) field trials to validate efficacy under real-world conditions, (ii) toxicity studies on non-target organisms, and (iii) investigations into the long-term stability of the nanoemulsions.

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Authors and Affiliations

1. Department of Medical Laboratory, College of Applied Medical Sciences, Prince Sattam bin Abdulaziz University, Al-Kharj, 11942, Saudi Arabia

   Mohamed A. El-Sakhawy & Abeer Ali El-Sherbiny Ateya

2. Department of Medicinal and Aromatic Plants, Desert Research Center, El-Mataria, Cairo, Egypt

   Mohamed A. El-Sakhawy

3. Department of Plant Protection, Desert Research Center, El-Mataria, Cairo, Egypt

   Mohamed A. Balah

Contributions

Conceptualization, Investigation, Methodology (A.A.E. Ateya), Analysis, investigation, methodology, writing ± original draft (M. A. El-Sakhawy), Data analysis, Methodology, writing and review (M. Balah).

Corresponding author

Correspondence to Abeer Ali El-Sherbiny Ateya.

Competing interests

The authors declare no competing interests.

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