Determination of the chemical composition of CEO. CEO is a complex compound that has active ingredients appropriate for natural herbicide products. GC–MS analyses identified 28 components constituting 99.72% of the total CEO. The major constituents were of the monoterpene class, namely geraniol (36.333% of total volatiles), trans-citral (17.881%), cis-citral (15.276%), citronellal (8.991%), β-citronellol (4.991%), and citral diethyl acetal (4.603%) (Table 2). The chemical composition of CEO as determined here is in agreement with the prior report of Nakahara, et al. 25, which found the major chemical constituents of essential oil from C. nardus to be geraniol (35.7%), trans-citral (22.7%), cis-citral (14.2%), geranyl acetate (9.7%), citronellal (5.8%), and citronellol (4.6%). Meanwhile, Timung, et al. 26 reported the main compounds of the oil from leaves of Java citronella (C. winterianus Jowitt) as citronellal (55.24%), geraniol (26.29%), and citronellol (13.41%). However, the variation in these reported chemical compositions can be attributed to several influencing factors such as species, harvest stage, genetic differences, climatic and environmental conditions, and the extraction method.
Table 2
Chemical composition of EO from citronella leaves.
Number | Class | Constituents | RTa min | % |
1 | Monoterpene | Camphene | 6.144 | 0.15 |
2 | | Methyl heptenone | 6.760 | 0.917 |
3 | | β-myrcene | 6.83 | 0.048 |
4 | | Octanal | 7.024 | 0.062 |
5 | | Limonene | 7.488 | 0.135 |
6 | | 2,6-Dimethyl-5-heptenal | 7.876 | 0.128 |
7 | | 4-Nonanone | 8.179 | 0.987 |
8 | | (-)-Linalool | 8.621 | 1.175 |
9 | | Citronellal | 9.479 | 8.991 |
10 | | trans-Caran-4-one | 9.905 | 1.11 |
11 | | Dacanal | 10.24 | 0.406 |
12 | | β-Citronellol | 10.607 | 4.991 |
13 | | cis-Citral | 10.833 | 15.276 |
14 | | Geraniol | 11.065 | 36.333 |
15 | | trans-Citral | 11.27 | 17.881 |
16 | | Citronellyl acetate | 12.312 | 0.371 |
17 | | Eugenol | 12.441 | 0.276 |
18 | | Geranyl acetate | 12.722 | 1.841 |
19 | | Neral dimethyl acetal | 13.169 | 2.328 |
20 | | Caryophyllene | 13.358 | 0.767 |
21 | | Citral diethyl acetal | 13.45 | 4.603 |
22 | Sesquiterpene | Humulene | 13.795 | 0.113 |
23 | | (-)-Germacrene D | 14.13 | 0.085 |
24 | | α-Cadinene | 14.518 | 0.188 |
25 | | Cadinene | 14.605 | 0.069 |
26 | | Elemol | 14.923 | 0.081 |
27 | Oxygenated sesquiterpene | Caryophyllene oxide | 15.403 | 0.377 |
28 | | (-)-α-Cadinol | 16.034 | 0.034 |
| Monoterpene | | | 98.776 |
| Sesquiterpene | | | 0.536 |
| Oxygenated sesquiterpene | | | 0.411 |
| Total | | | 99.72 |
Optimization of HLB values. Effect of Smix on droplet size, PI, and zeta potential value of CEO nanoemulsion. Initially, O/W nanoemulsion formulations were prepared using the high-energy technique from Smix with a range of HLB values. Ensuring an appropriate HLB value is essential for the formulation of a stable nanoemulsion. Droplet size and PI were considered when choosing the optimal formulation. A previous study has shown HLB values of 8–15 to be suitable for this type of O/W emulsion 17,27; accordingly, this study investigated the effect of Smix with HLB values in the range of 9-14.9. As shown in Fig. 1 and Table 3, nanoformulations with these HLB values consistently produced a mean droplet size below 200 nm, with the average droplet size decreasing as HLB value increased from 9 to 14. The smallest droplet size (78.8 nm) was obtained when using Smix at HLB 14. In general, increasing the Tween 60 fraction decreased droplet size; however, the formulation at HLB 14.9, which lacked a Span 60 fraction, exhibited increased droplet size (82.2 nm). This is consistent with prior observations that a nanoemulsion formulated with a surfactant mixture disperses and solubilizes better than one using only a single surfactant 27–29.
After preparation, the formulations made with Smix at HLB 13-14.9 appeared translucent with a blue tint and were without separated phase, flocculation, or coalescence. On the other hand, nanoemulsions at HLB 9–12 showed a separated phase. Accordingly, the optimized citronella oil-based nanoemulsion formulation was produced using Smix at HLB 14. Agrawal, et al. 27 also formulated citronella oil into an O/W nanoemulsion by a high-energy method (ultrasonic processor) using Tween 80 and Span 80 as the surfactant mixture. They likewise obtained a minimum droplet size at HLB 14.
PI indicates the homogeneity of a nanoemulsion. The nanoemulsions produced here at HLB 9-14.9 had respective PI values of 0.261, 0.276, 0.269, 0.302, 0.286, and 0.307.
In the present study, zeta potential was not considered as a criterion for selecting the optimal nanoemulsion. Table 3 lists the zeta potential values obtained for CEO nanoemulsions with HLB 9-14.9. A zeta potential of > + 30 or < -30 mV confirms that the nanoemulsion is stable, representing a high energy barrier toward the coalescence of dispersed droplets. However, this threshold is based on experiments and is not the only indicator for predicting nanoemulsion stability. The low zeta potential values in this work might be attributable to the use of nonionic surfactants (Tween 60 and Span 60) 30.
Selecting a suitable Smix is very necessary as it influences droplet characteristics. As the nanoparticle formulation with the smallest diameter was produced from Tween 60 (91.2%) and Span 60 (8.8%), the rHLB value for CEO was determined as HLB 14. This formulation was selected for evaluation in further experiments.
Table 3
Particle diameter, PI, and zeta potential of CEO-based nanoemulsions. The optimized formulation was produced at HLB 14. Means ± standard deviations. Means with different letters within a column are significantly different (p < 0.05).
HLB | Particle diameter (nm) | PI | Zeta potential (mV) |
9 | 151.8 ± 0.6 a | 0.242 ± 0.006 b | -37.22 ± 0.33 e |
10 | 133.2 ± 1.4 b | 0.240 ± 0.006 b | -23.34 ± 0.87 cd |
11 | 125.6 ± 1.0 c | 0.236 ± 0.007 b | -23.16 ± 1.02 c |
12 | 121.4 ± 0.8 d | 0.243 ± 0.007 b | -25.24 ± 1.29 d |
13 | 87.9 ± 0.7 e | 0.265 ± 0.006 a | -16.76 ± 0.85 a |
14 | 78.8 ± 0.5 g | 0.276 ± 0.005 a | -19.70 ± 1.11 b |
14.9 | 82.8 ± 0.2 f | 0.277 ± 0.004 a | -18.38 ± 1.11 ab |
Characterization of the selected formulations. Effect of Smix concentration on nanoemulsion droplets. In the present study, Smix (HLB 14) concentration was varied over a range of 0.5% − 4%., while that of the dispersed phase (CEO) was held constant at 2%. The effect of Smix concentration on nanoemulsion droplet size, PI, and zeta potential value is summarized in Table 4. Increasing the proportion of Smix from 0.5–2% significantly reduced droplet size (from 117.3 to 78.8 nm); however, further increasing Smix to 4% increased droplet size (85.7 nm). Notably, the nanoemulsion prepared with a Smix concentration of 2% appeared to be translucent, but that using 4% Smix became more turbid. Carpenter and Saharan 17 previously reported that increasing surfactant fraction resulted in an insufficiency of the interfacial sites that lead to micellization of surfactant molecules in the water phase, which generated increased turbidity of emulsion.
Greater Smix concentration also influenced other characteristics such as PI and zeta potential (Table 4); for example, when increasing Smix concentration from 0.5–4%, the PI value also increased from 0.1472 to 0.3030.
Table 4
Particle diameter, PI, and zeta potential of CEO-based nanoemulsions incorporating different concentrations of Smix at HLB 14. Means ± standard deviations. Means with different letters within a row are significantly different (p < 0.05).
Smix concentration (%) | 0.5 | 1 | 2 | 4 |
Particle diameter (nm) | 117.3 ± 0.5 a | 88.6 ± 0.7 b | 78.8 ± 0.5 d | 85.7 ± 0.8 c |
PI | 0.1472 ± 0.018 d | 0.2078 ± 0.007 c | 0.2762 ± 0.005 b | 0.3030 ± 0.004 a |
Zeta potential (mV) | -22.86 ± 0.94 d | -9.31 ± 0.52 a | -19.70 ± 1.11 c | -16.53 ± 0.53 b |
Surface tension and pH value. The optimal formulation of CEO-based nanoemulsion, namely that having the smallest particle diameter, was further evaluated and characterized. It exhibited a surface tension of 31.67 mN/m, which will assist in easy delivery of the essential oil to plants as an agrochemical due to possessing higher wetting, spreading, and penetrating properties 31. Solution pH was evaluated as a stability indicator, as most pesticide nanoemulsions degrade with an alkaline solution or a high pH value 32. In the present study, the selected nanoemulsion had a mildly acidic pH value (pH 5.1), indicating that it could be stable.
Morphology. To confirm droplet size in the selected O/W nanoemulsion, droplet shapes were investigated through transmission electron microscopy (TEM) images, of which representative examples are shown in Fig. 2. The morphology of nanodroplet clusters in the nanoemulsion was spherical, with an interior gray part (oil) surrounded by a black ring (surfactant). In the pictures, nanodroplet size ranged 50–120 nm and correlated to the average droplet size as determined by the DLS technique. Similarly, Kumari, et al. 33 reported TEM analysis of a thymol nanoemulsion to show spherical droplets of 80–150 nm.
Storage stability of the CEO nanoemulsion. The optimal formulation (HLB 14) was investigated with regard to the effect of temperature and storage time on stability of the CEO nanoemulsion. The chosen formulation was stored at temperatures of 4, 25, and 45°C, and the droplet size, PI, and zeta potential value were assayed every seven days over a total storage period of 28 days. The changes in droplet size during storage at each tested temperature are given in Table 5. At a temperature of 4°C, the mean droplet size decreased over time; however, emulsions stored at 45°C exhibited greater droplet size, increasing from 78.8 to 217.5 nm after 14 days. Furthermore, separation of phases was seen after 21 days at 45°C. Borba, et al. 34 previously described that centrifugal force and high temperature could accelerate Brownian motion. Hence, droplets may move close to each other, causing an opportunity for destabilization with enlarged droplet size. However, while separation was observed after storage for 21 days at 45°C, the nanoemulsion remained stable (< 200 nm) after just 7 days at that temperature. Meanwhile, for the nanoemulsion stored at room temperature, droplet size changed only slightly over the full 28-day storage period (Table 5). These results indicate that temperature has substantial influence on nanoemulsion droplet size and related properties, including PI. The size distribution decreased with storage time, reaching values below 0.276 in all treatments. These results agree with Teng, et al. 35, which reported that storage temperature and time duration are the most important factors influencing nanoemulsion stability. Namely, the nanoemulsion gradually oxidizes during storage, and the degree of oxidation increases with storage time and temperature. Lipid oxidation might change the interfacial composition of the nanoemulsion, causing the emulsifier to rearrange and desorb at the interface and hence reducing the stability of the emulsion system. Eventually, the stored nanoemulsions break up, which causes CEO to be released from the nanoemulsion. Particle size is a significant parameter in ensuring the physical stability of nanodroplet formulations under storage.
Table 5
The effect of storage temperature and duration on particle size, PI, and zeta potential of CEO-based nanoemulsions. Means ± standard deviations. Means with different uppercase letters are significantly different (p < 0.05) within the row. Means with different lowercase letters are significantly different (p < 0.05) within the column (same parameter and day for different temperatures).
Duration (days) | 0 | 7 | 14 | 21 | 28 |
Particle size (nm) |
4°C | 78.8 ± 0.5 Aa | 59.8 ± 0.5 Bc | 57.2 ± 0.2 Cc | 57.9 ± 0.5 Cb | 57.9 ± 0.4 Cb |
25°C | 78.8 ± 0.5 Da | 73.7 ± 0.7 Eb | 86.9 ± 0.3 Cb | 117.0 ± 1.0 Ba | 139.8 ± 1.8 Aa |
45°C | 78.8 ± 0.5 Ca | 105.7 ± 0.8 Ba | 217.5 ± 0.8 Aa | - | - |
PI |
4°C | 0.276 ± 0.005 Aa | 0.190 ± 0.006 Cb | 0.197 ± 0.005 Ca | 0.190 ± 0.009 Ca | 0.224 ± 0.008 Ba |
25°C | 0.276 ± 0.005 Aa | 0.215 ± 0.004 Ba | 0.172 ± 0.009 Cb | 0.103 ± 0.012 Db | 0.107 ± 0.014 Db |
45°C | 0.276 ± 0.005 Aa | 0.132 ± 0.015 Bc | 0.109 ± 0.016 Cc | - | - |
Zeta potential (mV) |
4°C | -19.70 ± 1.11 Ca | -16.60 ± 1.66 Ba | -24.46 ± 0.75 Db | -16.29 ± 1.12 Bb | -12.24 ± 0.93 Aa |
25°C | -19.70 ± 1.11 Ba | -21.20 ± 1.83 Bb | -19.79 ± 0.52 Ba | -13.92 ± 1.93 Aa | -12.75 ± 0.53 Aa |
45°C | -19.70 ± 1.11 Aa | -20.32 ± 1.08 Ab | -19.41 ± 0.68 Aa | - | - |
Herbicidal activity of CEO nanoemulsion. Seed germination and seedling growth. The effects of nanoemulsions formulated at various HLB values and concentrations on germination and seedling growth of E. crus-galli were investigated using the Petri dish test. Nanoemulsion HLB ranged from 9 to 14.9, and the solutions had different droplet sizes. All were found to affect germination percentage and root and shoot length in the tested weed, those parameters decreasing with increasing nanoemulsion concentration; in addition, dose-responses relationship with HLB value were observed. Seven days after treatment, the nanoemulsions at HLB 13-14.9 presented a remarkable effect on seed germination (Fig. 3). Overall, the effect of the nanoemulsion at HLB 14, which featured the smallest particle size (78.8 nm), was most excellent relative to solutions prepared with other HLB values. As shown in Fig. 3, treatment with HLB 14 solution at the highest tested concentration (800 µL/L of CEO) completely inhibited the germination of E. crus-galli seeds. However, treatment with the HLB 13 solution (droplet size 87.9) at the same concentration also showed an inhibition of germination that was not significantly different from that obtained with HLB 14. Meanwhile, nanoemulsions at HLB 9–11 exhibited the lowest inhibitory effect on germination of E. crus-galli seeds, consistent with these solutions having droplet sizes above 100 nm (125.6–151.8 nm).
In addition to affecting seed germination, the CEO nanoemulsions also impacted seedling growth of E. crus-galli (Fig. 4–5). Physical evaluation of shoot and root lengths revealed both to be reduced by nanoemulsion treatment across the range of tested HLB values. Shoot length exhibited different degrees of inhibition (Fig. 4), with the maximum shoot length being recorded in the control (data not shown). Results for root length also differed across tested HLB values and exhibited a dose-dependent response (Fig. 5). In short, CEO nanoemulsions have a dose effect on E. crus-galli seed germination and seedling growth, with that at HLB 14 having the highest inhibitory potential.
These results are in agreement with previous research indicating that nanoemulsions at similar concentrations have greater inhibitory potential on seed germination and seedling growth than ordinary emulsions (> 200 nm) due to their tiny particle size. In addition to larger particle size, other factors that constrain the allelochemical potential of oil components include evaporation and oxidation 1,31. Accordingly, nanoemulsions have been formulated to improve the properties of essential active chemicals, ensuring their effective release and rapid interaction with plant cells after application on weed seeds or leaf surfaces 31,36,37. In the present study, CEO was determined to consist of 95% monoterpenes, biologically active compounds that have demonstrated inhibitory potential against seed and seedling growth. Several prior reports have found monoterpenes to show various allelopathic effects on seed germination, with hydrocarbons being minor inhibitors compared to oxygenated monoterpenes 38,39. Our results confirm that the nanoemulsion developed from CEO, Tween 60, and Span 60 by the high-energy method has enhanced potential to inhibit germination and seedling growth of E. crus-galli.
The inhibitory effects of this nanoemulsion on the tested seeds could be attributed to the minor and major compounds in CEO, the concentration of CEO, and the particle size of the nanoemulsion. To further study the phytotoxicity of the nanoemulsion on E. crus-galli, the optimal solution with HLB 14 was utilized in experimental assays.
Seed imbibition. Seed imbibition is the initial step in the process of seed germination. The effect of the nanoemulsion formulation at HLB 14 was investigated with respect to water adsorption by the tested seeds over 12, 24, and 36 h. For a given concentration of treatment, the percentage of seed imbibition increased with imbibition period. In the first 12 h of imbibition, control seeds soaked in water showed the highest water adsorption, which was not significantly different from that obtained with Smix solution alone or most treatment solutions (100, 200, and 400 µL/L of CEO) (Fig. 6). However, treatment with the highest concentration of CEO (800 µL/L) consistently resulted in the lowest seed imbibition percentage across the time course. Moreover, at 36 h, the percentage of seed imbibition decreased with increasing concentrations of CEO.
These results are in line with observations by Teerarak, et al. 40 from treatment of E. crus-galli seeds with Aglaia odorata Lour. essential oil, in which the inhibitory effect on imbibition was elevated for increased concentrations of oil.
α-Amylase activity. Figure 7 presents the effect of the nanoemulsion at HLB 14 on seed germination in relation to α-amylase activity and carbohydrate degradation. In the germination process, α-amylase acts to digest starch into small organic molecules, thereby producing the nutrients and energy needed for germination 41. To test whether a decrease in α-amylase activity mediated CEO-induced seed germination inhibition, the effect of the CEO nanoemulsion at concentrations of 100, 200, 400, and 800 µL/L on α-amylase activity was investigated. Overall, CEO-based nanoemulsions reduced α-amylase activity of the tested weed seeds relative to the water control. After 12 h, the nanoemulsion-treated samples were not significantly different from the control; however, after 24 and 36 h, α-amylase activity was significantly decreased in a dose-dependent manner. Mainly, the highest tested CEO concentration (800 µL/L) favorably inhibited α-amylase activity, which is consistent with the results of the imbibition assay (Fig. 6). Poonpaiboonpipat, et al. 42 similarly investigated the mechanism by which Cymbopogon citratus EO inhibits seed germination. They reported that the α-amylase activity of E. crus-galli seeds treated with the oil was decreased. Similarly, Laosinwattana, et al. 43 investigated the inhibitory effect of Tagetes erecta L. EO on α-amylase activity of E. crus-galli seeds using an emulsifiable concentrate of the EO (EC-EO). Their report indicated that the EO could inhibit α-amylase activity in a dose-dependent manner, with the highest tested concentration (2000 µL/L of EC-EO) exhibiting a significantly outstanding decrease of seed α-amylase activity at 48 h.
The results of the present study show that inhibition of α-amylase activity is one of the herbicidal activities of CEO nanoemulsions. This effect could in turn cause inhibition of seed germination and seedling growth due to starch not being degraded into small molecules to fuel growth and development.