Preparation of nanoemulsions containing herbal derivatives: a dynamic light scattering analysisAbstract
Citral and quercetin are, respectively, a monoterpene and a flavonoid of great interest for cosmetics. They have impaired water solubility, and the development of nanoemulsions is a suitable strategy to overcome this main issue. In this study, citral was used as the oily phase to nanoemulsify quercetin through a low-energy method. The nanoemulsions were prepared with deionized water or glycerol, and the best results were reached with polysorbate 80 and water, being observed a size diameter of around 20 nm and a relatively monodisperse distribution. Quercetin was capable of reducing droplet size when compared to citral, a mandatory component for classifying this colloid as a nanoemulsion. Up to 65ºC, there was no main difference in droplet size. Therefore, this study provides suitable prototypes of citral/quercetin nanoemulsion that can be further used in cosmetic applications through an eco-friendly method of preparation.
Keywords:
geranial; neral; quercetin; hydrodynamic radius
Resumo
Citral e quercetina são, respectivamente, um monoterpeno e um flavonoide de grande interesse para cosméticos. Eles têm limitada solubilidade em água e o desenvolvimento de nanoemulsões é uma estratégia apropriada para sanar esse problema. Nesse estudo, o citral foi utilizado como fase oleosa para nanoemulsificar quercetina através de um método de baixa energia. As nanoemulsões foram preparadas com água deionizada ou glicerol, e os melhores resultados foram observados com polisorbato 80 e água, sendo observado diâmetro médio em torno de 20 nm e distribuição relativamente monomodal. A quercetina foi capaz de reduzir o tamanho de gotícula quando comparada ao citral. Até 65º C, não foi observada diferença no tamanho de gotícula. Portanto, esse estudo permitiu a obtenção de protótipos de citral/quercetina que podem ser futuramente utilizados e cosméticos através de um método de preparação amigável ao meio ambiente.
Palavras-chave:
geranial; neral; quercetina; raio hidrodinâmico
1. Introduction
Quercetin (C15H10O7) is a flavonoid widely distributed in plants of several species. It has a cetocarbonyl group and a basic C1 oxygen. A phenolic hydroxyl and double bonds turn quercetin into a powerful antioxidant (Ulusoy and Sanlier, 2020). It is a yellow solid with a bitter flavor, insoluble in water and soluble in alcohol, lipids, aqueous alkaline solutions, and glacial acetic acid (Singh et al., 2021; Aghababaei and Hadidi, 2023).
Absorption, solubility, and biological effects may be influenced by glycosyl groups bonded to quercetin, thus generating analogues (Aghababaei and Hadidi, 2023). It is classified according to the Biopharmaceutical Classification System as class IV due to low solubility and low permeability (Rao et al., 2024).
Another type of natural product widely distributed in plants is citral (C10H16O). It is a monoterpene found in essential oils and it is constituted by acyclic isomers geranial and citral. It also has antioxidant capacity, being also used as a cosmetic ingredient due to this property, among others (Saddiq and Khayyat, 2010; Lu et al., 2018; Capetti et al., 2021). Intrinsic low water solubility is observed for essential oil components, and therefore, it is a technological challenge to turn some natural products into viable aqueous products, such as those desirable for cosmetics (Pereira et al., 2021; Bajerski et al., 2016; Santos et al., 2019).
Nanotechnology is a multidisciplinary area that includes the bioactive compounds delivery systems, comprehended between 1 – 1000 nm. Growing interest in nanocosmetics is observed, attracting consumers due to several advantages compared to bulk materials (Gupta et al., 2022). These advantages include high stability and proper penetration in skin due to high surface/volume area (Yadwade et al., 2021).
A type of nanotechnology-based system of application in cosmetics is called nanoemulsion. According to Singh et al. (2017), the nanoemulsions are colloidal dispersions with internal phase droplets on a nanometric scale (20 - 500 nm). Due to low droplet diameter, they are macroscopically transparent or translucid (translucid < 200 nm > opaque) and present a bluish reflect due to the Tyndall effect (McClements, 2012; Santos et al., 2019).
The conventional nanoemulsions have often three components: an organic phase (oil), an external phase (water), and surfactant (s). Since they are not thermodynamically stable, energy is needed for nanoemulsification (McClements; Jafari, 2018).
According to Solans and Solé (2012), low-energy methods are preferable for nanoemulsification instead of high-energy methods (e.g., ultrasonication, high-pressure homogenization, and others). This is since most of the energy is dissipated as heat. However, highly disruptive forces can help if the researcher prefers high-energy methods (Gupta et al., 2016; Singh et al., 2017; Pereira et al., 2021).
The organic phase can be any oil immiscible in the external phase, in the case of the most common oil in water nanoemulsions. Essential oils and their components can often be used for this purpose. The external phase most commonly used is water; however, miscible components in this medium can also be used, such as glycerol and others. (Jaiswal et al., 2015; McClements; Jafari, 2018).
The utilization of low-energy methods capable of inducing phase inversion is widely used and can generate fine droplets that can be scaled up (Solans and Solè, 2012). Tendencies of the market are also changing, especially regarding phytocosmetics. Nanotechnology, therefore, can help achievement of novel products with high performance. In this context, this study aims to investigate the influence of parameters for nanoemulsification of natural products of great interest for cosmetics, being from two classes: flavonoid and monoterpene, providing prototypes for further investigation of phytocosmetic potential.
2. Methods
2.1. Materials
Quercetin was obtained from Interlab (São Paulo, Brazil) and citral from Quinari (São Paulo, Brazil).
2.2. Preparation of nanoemulsions
Aqueous dispersions of non-ionic surfactants (external phase) were prepared by adding the surfactant in deionized water and stirring until homogenization (2 min). An ethanolic solution of natural products (citral/quercetin) was prepared to achieve the organic internal phase. Thus, the organic phase was titrated dropwise through aqueous dispersion at a 1:10 ratio under vortex stirring for 3 min. The obtained systems were stored in screw top vials for further characterization.
4.2. Factors of influence in the nanoemulsification
4.2.1. General composition
The nanoemulsions were prepared at a final composition as follows: 0.4% (w/w) of quercetin and 4.0% (w/w) of citral. According to the supplier, citral was constituted by 45.1% (w/w) of neral and 52.6% (w/w) of geranial.
4.2.2. Surfactants
The nature of employed non-ionic surfactants was varied by using polysorbate 20 or polysorbate 80. The final concentration of surfactants ranged as follows: 2, 5, 10, 15, and 20% (w/w).
4.2.3. External phases
The external phase was varied by using deionized water, glycerin, aqueous dispersion of surfactants, or glycerin dispersion of surfactants.
4.3. Characterization
4.3.1. Macroscopical evaluation
The nanoemulsions were characterized according to the presence of the Tyndall effect, iridescence, creaming, sedimentation, and phase separation. Those with satisfactory characteristics were submitted to centrifugation at 3000 rpm/30 min.
4.3.2. Dynamic light scattering (DLS)
A Zetasizer Advancer Pro (Blue) (Malvern, UK) was used for size distribution evaluation. The transparent sample (s) were directly placed into a glass cuvette, and turbid samples were diluted in deionized water (1:20) before analysis. The refractive index of the citral was selected for the encapsulated material, and the equilibrium time was 30s. A sample without quercetin was prepared at the equivalent composition of a chosen nanoemulsion to evaluate the influence of quercetin on the nanoemulsification. The formulations were characterized after 10min and 24h of preparation. The zeta potential was evaluated by electrophoretic light scattering (ELS) under the same conditions described for DLS analysis. All analyses were performed in triplicate.
4.3.3. Linear ramp of heating
A linear increase in the temperature was performed by using a programmed ramp at a 5ºC/cycle in the range of 25 to 75 ºC. Equilibrium time was 30 s with return to default temperature, and the same conditions for overall dynamic light scattering analysis.
3. Results
At 2, 5, 15%, and 20% of polysorbate 20 (Figure 1), it was possible to observe different signals of instability, such as sedimentation and creaming. Also, a milky appearance was observed for these systems. In the nanoemulsions prepared with polysorbate 80, a tendency for lower turbidity with indicative of nanodroplets through a bluish reflect. However, only at a higher amount of polysorbate 80 was it possible to reach transparency.
Nanoemulsions prepared with quercetin and citral. Upper panel prepared with polysorbate 20 (from left to right, surfactant to oil ratios of 9:1, 8:2, 7:3, 6:4, and 5:5). Lower panel prepared with polysorbate 80 (from left to right, surfactant to oil ratios of 9:1, 8:2, 7:3, 6:4, and 5:5).
Nanoemulsions are kinetically stable systems with a transparent or translucent aspect with a characteristic bluish reflect. In some cases, opacity due to a high contrast between internal and external phases can be observed (Ling, 2022). Creaming and sedimentation are undesirable and the low droplet diameter avoids gravitational separation. However, destabilization due to Ostwald ripening may occur if one of the components of the internal phase has limited solubility in the external phase, therefore migrating from smaller to bigger droplets and increasing size (McClements and Jafari, 2018).
Lower size was observed for the nanoemulsions prepared with aqueous solution, being the nanoemulsion prepared with 20% of polysorbate 80 the one with the lowest size diameter (16.2 ± 0.2 nm) and lower PdI (0.4396 ± 0.0660) (Table 1). However, high PdI was observed for the nanoemulsion prepared with polysorbate 20 at 15% on the day of preparation, being associated with a higher percentage (65%) of high diameter droplets (4~90.0 nm).
Nanoemulsions prepared with glycerol presented either a size close to 200 nm or a high PdI during the analyzed period. Regarding the most intense peak (Peak 1), a higher abundance of low nanodroplets was observed for the nanoemulsion prepared with aqueous solution and 20% of polysorbate 80 (~15 nm, 74 – 83.7%). Therefore, the system with this surfactant was chosen for additional investigations.
The droplet size of the formulations was statistically different (p < 0.05), as confirmed by Tukey's post hoc test. Regarding the polydispersity index, most pairwise comparisons showed statistically significant differences between formulations. Only the comparisons between NE Aqueous Solution T80 20% and NE Glycerol Solution T80 20% (p = 0.0915) and between NE Aqueous Solution T80 20% and NE Glycerol Solution T80 15% (p = 0.6111) were not statistically significant. These results suggest that both the type of dispersing phase and the concentration of polysorbate 80 affected the uniformity of droplet size distribution across the nanoemulsions.
The effect of quercetin on droplet size in nanoemulsions was previously reported. Karadag et al. (2013) observed that higher quercetin content tends to increase droplet size but also improves physical stability. This may be due to the hydrophobic nature of quercetin, which in greater amounts can persist in a stable supersaturated state, delaying crystallization. In contrast, lower concentrations may favor early crystal formation and phase separation. Therefore, despite the increase in droplet size, quercetin may contribute to stability by modulating nucleation and precipitation during storage (Karadag et al., 2013).
Similar findings were described by Hsieh et al. (2025), who also reported that increasing quercetin content in nanoemulsions led to larger droplet sizes and a broader size distribution, possibly due to its preferential incorporation into the oil phase and its effect on interfacial organization (Hsieh et al., 2025). Although the present study did not vary quercetin concentration, these findings help contextualize the observed droplet size and stability profiles. Here, nanoemulsions were formulated using fixed quercetin content but varying surfactant concentrations (15% and 20%). The aqueous formulation with 20% of polysorbate 80 (Tween 80) showed smaller droplet size, lower polydispersity index, and greater physical stability over time, suggesting that the surfactant concentration played a more critical role in stabilizing the system. This supports the notion that both the interfacial composition (e.g., surfactant level, quercetin concentration) may influence the final droplet characteristics and stability of nanoemulsions.
Figure 2 shows the size distribution graphs with and without a fluorescence filter. Overall, no main difference was observed, especially for low-diameter peaks (< 20 nm). An exception was observed due to a slightly higher size diameter for the peak around 100 nm after one day of preparation, when the fluorescence filter was applied. Some fluorescence compounds can interfere with the analyses, since DLS is an indirect measurement of the hydrodynamic radius (Fu et al., 2020). However, it does not compromise the practical application of these nanoemulsions in cosmetics. No main differences were observed in the present study.
Superposition of size distribution graphs of the nanoemulsion prepared with aqueous solution and 20% polysorbate 80 without quercetin at day 0 (upper panel) and after 1 day of storage (lower panel). Graphs obtained without fluorescence filter (blue) and with fluorescence panel (red).
The nanoemulsion prepared with aqueous phase and 20% of polysorbate 80 was prepared without quercetin to evaluate the influence of this flavonoid. An increase of mean droplet diameter to 41.1 ± 9.1 nm was observed without main alteration of PdI (0.430 ± 0.03). Also, the low size peak (< 20 nm) had a decrease in the intensity (49.1 ± 1.4%). According to the Chemspider Plattform® Citral has the following molecular/physicochemical in silico properties: logP = 3.17, molar volume = 177.8 ± 3.0 cm3 and surface tension = 27.1 ± 3.0 dyne/cm, while quercetin has as follows: logP = 2.08, molar volume = 168.0 ± 3.0 cm3 and surface tension = 114.9 ± 3.0 dyne/cm. Therefore, probably a slightly improved release to the external phase than citral, saturating the external media without providing Ostwald ripening and also adherence to the interface may contribute to a lower size of quercetin/citral nanoemulsions.
Otherwise, the composition prepared with aqueous phase and 20% of polysorbate 80 without citral as the oil phase showed a mean droplet size (nm) of 9.7 ± 0.11, an average polydispersity index (PdI) of 0.15 ± 0.04, and an average zeta potential of -7.6 ± 0.31 mV after preparation. Additionally, after 24h of preparation, the formulation without citral showed an average droplet size of 9.7 ± 0.06 nm, 0.12 ± 0.06 of PdI, and -0.14 ± 0.51 mV of zeta potential.
As expected, the absence of a clearly defined oil has been demonstrated to exert a significant influence on the generation of the colloidal system. It is possible to observe a population of droplets in the range of 10 to 20 nm, characteristic of micellar solutions (McClements, 2012). This is due to the excess polysorbate 80 acting as a solubilizing agent rather than a stabilizer of the nanometric droplet interface. It is hypothesized that quercetin will dissolve in water, facilitated by the presence of polysorbate 80 micelles and ethanol.
Droplet size distribution after a linear ramp of temperature (25 to 75 ºC). (A) size, diameter, and (B) polydispersity index of the nanoemulsion with polysorbate 80 in aqueous solution at 20%.
According to Prakash and Vadivel (2020), the ultrasonication of citral with polysorbate 80 at a 1:1 ratio induced a size around 20 nm and PdI of around 0.45, being the results similar to those obtained in this study. However, in the present study, we opted for a low-energy method, which is an advantage in comparison to high-energy methods (Solans and Solè, 2012).
The linear ramp of temperature of NE Aqueous Solution T80 20% (Figure 3) showed a significant increase in size from 15.8 ± 0.06 nm at 25 ºC to 291.0 ± 23.3 nm at 75ºC (p < 0.01); however, an overall maintenance of size was observed in the range of 25 ºC to 65°C. PdI presented a tendency for increase, reaching a maximum at 55 ºC (p<0.01). Reduction after this temperature may be due to destabilization artifacts generated during the enhancement of temperature. These findings highlight the potential of low-energy nanoemulsification using polysorbate 80 to produce stable quercetin-loaded systems with desirable physicochemical properties, reinforcing its applicability for thermally stable delivery formulations.
Acknowledgements
The authors would like to thank CNPQ for the financial support (process number 317405/2021-7).
Data Availability Statement
The entire data set that supports the results of this study was published in the article itself.
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Edited by
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Editor:
Takako Matsumura Tundisi
