3.4.2. Changes in Particle Size of LEO-NEs during the Storage Period

Figure 3b demonstrates the changes in the mean droplet of LEO-NEs for 1 week at a storage temperature of 25 ◦C. The particle size of the LEO-NEs changed little during a week, ranging from 62.96 nm to 64.60 nm.

**Figure 3.** Physicochemical Properties and Stability of LEO-NEs: (**a**) particle size distribution and transmission electron microscopy (TEM) of droplets of LEO-NEs; (**b**) effect of storage time on particle size of LEO-NEs; (**c**) antioxidant activity comparison between LEO-NEs and LEO.

#### 3.4.3. Antioxidant Activity of LEO-NEs

The DPPH and ABTs assays were used to measure the free-radical-scavenging potential to compare the difference between LEO-NEs and EO at the same concentration (0.05 g/mL). It has been shown that EOs exhibit antioxidant activities due to large amounts of polyphenol compounds. Figure 3c shows that the antioxidant activity of the LEO-NEs was higher than that of LEO with the same concentration, whereby the inhibition of DPPH free radicals by LEO-NEs (57.61%) was much better than that by LEO (8.74%), but the inhibition of ABTs free radicals by LEO-NEs (31.74%) was similar to that by LEO (30.61%).

#### **4. Discussion**

Coinciding with the reports by other Hirai et al. [25], Aguilar et al. [26], Perdones et al. [27] and Campolo et al. [28], limonene was the most abundant component in LEO while its content may vary. LEO was rich in constituents with monoterpene structure (limonene, α-pinene, etc.) which have been proven to possess antioxidant activity [29]. For example, limonene was shown to prevent neuronal suffering [30], oxidative stress on lymphocytes, and mitochondrial dysfunction [29] through its antioxidant activity. In addition, LEO components present in other studies were not detected in this experiment such as β-phellandrene [25], camphene, and sabinene [26]. Differences in LEO composition may be due to differences in geographic location, environmental factors, plant age, developmental stage, harvest time, extraction site, and extraction method [31].

In this study, LEO-NEs were prepared by the ultrasonic method using STmix as an emulsifier. The influence of each factor was studied by SFEs. Firstly, when the HLB values of STmix ranged from 8–14, the mean droplet size of NEs was less than 200 nm, while NEs could not be formed when HLB was 15. The large range of suitable HLB values means that many kinds of surfactants can be used to prepare LEO-NEs. Tween-80 was not suitable for this experiment; however, it was also used to make lemon LEO-NEs in other experiments. Mossa et al. [13] reported that the droplet size of LEO-NEs was 131.9 nm, while the particle size of the LEO-NEs was 181.5 nm in the study of Yazgan [32]. Although these studies were able to form NEs with Tween-80, the particle sizes were larger than 100

nm. Furthermore, the droplet diameter of LEO-NEs was 91 nm [33] and 135 nm [34] when produced with Tween-80 using the high-pressure homogenizer method. These results indicate that different essential oil components and different emulsification methods may lead to different particle sizes when constructing NEs.

When the concentration of essential oil was 0.05–0.1 g/mL, the particle size increases with the increase in concentration of essential oil, indicating its greater impact on particle size. However, previous studies showed that the particle size would not increase when the essential oil exceeds a certain amount if the concentration of surfactant micelles remains not changed, because of a saturation with lemon oil, whereby any further lemon oil droplets added to the nanoemulsions would not dissolve [35]. This phenomenon did not occur in our experiments because the concentration range of LEO was not large enough. An increase in surfactant concentration can also lead to a decrease in particle size. The surfactants can affect inter-particle interactions in emulsions, whereby a the higher surfactant concentration results in weaker inter-particle interactions and smaller droplets formed [36]. The effect of surfactant concentration on mean particle size may be related to the surfactant dose required to cover the surface of the formed droplets, whereby self-emulsification would be more dependent on surfactant concentration [37]. In the process of ultrasonic preparation of NEs, the particle size did not decrease when the surfactant concentration increased to a certain amount. In addition, the dependence of the mean particle size on surfactant concentration did not depend strongly on storage time and temperature [12].

Ultrasonic cavitation is a feasible and energy-efficient method for preparing NEs, which offers improvements in terms of stability and decreases the Ostwald ripening rate. During the ultrasonication processes, soundwave energy causes cavities and sinusoidal pressure variations in the liquid–liquid interphase, resulting in a shockwave action on the particle surface and a reduction in particle size [38]. The particle size of nanoemulsions prepared with the ultrasonic method is generally determined by the sonication time and sonication power, but is insensitive to ultrasonication amplitude [39]. Understanding the dynamic routes is critical for reducing processing time and avoiding energy oversupply. When the ultrasonic time reached a certain value, the particle size reached the minimum. Increasing the ultrasonic time would not lead to a significant change in particle size. The increase in ultrasonic power led to a decrease and then increase in particle size, coinciding with the report of Kentish et al. [40]. In addition, Floris et al. [41] reported that high ultrasonic power may destroy bioactive substances.

The small size of the particles in NEs would result in less agglomeration or precipitation and higher stability of the system [42,43]. RSM was used to optimize NEs to obtain the smallest droplet size. The interaction between surfactant concentration and ultrasonic time had the greatest effect on particle size. However, the particle size did not decrease indefinitely, as it was limited by the ingredients of the essential oil. The optimal preparation conditions obtained by RSM were similar to those obtained by SFE, and the conditions predicted by RSM were relatively more precise.

Due to the mass transfer of oil molecules, droplets in NEs change from smaller droplets to larger droplets through an intermediate water phase, which is called Ostwald ripening. Ostwald ripening leads to droplet growth and phase separation [44]. From the TEM image and the particle size change during storage, particle diameter does not exceed 200 nm; hence, the ripening phenomenon was not serious in LEO-NEs. However, the TEM images revealed that the diameters of the particles in the nanoemulsion were not the same. The TEM image was similar to that presented by Kaur et al. [45] and Zhong et al. [46]. In previous studies, the structure of NE was presented a spherical substance consisting of several small spherical packets [46]. The particle size of LEO-NEs had the tendency to decrease in 1 week, possibly due to the EOs in the NEs undergoing a small amount of evaporation, thereby reducing the content of essential oil. In the study of Zhong et al. [46], there was a tendency for the particle size to increase with storage time, which may have been due to Ostwald ripening.

The prepared NE was not only stable but also had sustained-release activities. The study of antioxidant activities is essential as reflected in the reduction in reactive oxygen species (ROS) in the food and cosmetics industries. We could find that the encapsulation of essential oils in NEs helped to enhance their antioxidant activities when comparing the antioxidant activity of essential oils and NEs. DPPH scavenging activity refers to the ability to reduce the stable DPPH free radical to its reduced form DPPH-H [47]. ABTs scavenging activity refers to the ability to decolorize the radical cation (ABTS•+) [48]. Due to the different principles of determination, the two results are not necessarily related. The different methods employed to indicate antioxidant activity can comprehensively profile the antioxidant activities of LEO-NEs. According to a previous study [6], the DPPH radical scavenging activity and ABTs radical-scavenging activity of pure LEO were 32.85% and 41.57% respectively. These results are close to the antioxidant capacity of LEO-NEs in our study, but the composition and

determination method of the essential oil had an impact on the results. In addition, the antioxidant activity of LEO and LEO-NEs may be due to components in LEO with antioxidant activity. However, The antioxidant activity of LEO should not only consider the primary constituents [49]. The main antioxidant components in lemon essential oil need to be further studied.
