1. Introduction
Ethanol forms the base for traditional perfumes available on the market, since it is the solvent of hydrophobic aromatic substances. Depending on the contents of a fragrance composition, we distinguish between eau de parfum which contains 10–15% of a fragrance composition, eau de toilette which contains 5–10% of a fragrance composition, and eau de cologne which contains 3–5% of a fragrance composition [
1,
2]. Unfortunately, people prone to allergies or with sensitive skin, who use ethanol-based perfumes, may suffer from skin irritation and inflammation because ethanol is a solvent with a defined irritating potential [
3].
As an alternative to alcohol compositions, there are perfumes in a solid state (solid emulsions, gels, and pomades) and perfumed oils. However, their main disadvantage is that they leave greasy and slippery spots on the perfumed surfaces. Some examples of perfumed products are presented in the literature and they are based on a safe solvent like water, the so-called “alcohol-free perfumes” (emulsions, microemulsions, liposomes, and micelles) [
4,
5,
6,
7,
8,
9]. Yet from a technological perspective, the introduction of lipophilic systems to water without a cosurfactant (ethanol, among others) is very difficult, with respect to thermodynamic stability. It requires the use of solubilizers of essential oils which can be polyols (glycols and glycerin) or surfactants.
An interesting solution for the water-based perfumes without alcohol, seem to be nanoemulsions—liquid colloidal systems characterized by a high degree of dispersion (20–500 nm), consisting of aqueous and oil phases and a surfactant, sometimes with the addition of a cosurfactant. Similar to microemulsions, nanoemulsions increase the expiry date of many products, due to their resistance to sedimentation and creaming. An additional advantage over microemulsions is that they have a much lower amount of a surfactant (approx. 5–10%), which allows maintaining adequate stability of the system and it makes them safe for human body [
10,
11,
12,
13,
14]. Clear appearance, liquidity, and low viscosity, make nanoemulsions even more popular with the cosmetic industry. The examples of nanoemulsion cosmetics found on the market are anti-UV hair spray by Korres, Nanocream by Sinerga, Nanogel by Kemira, Vital Nanoemulsion A-VC serum by Marie Louise, Bepanthol Ultra face cream by Bayer, or face cleanser NanoVital by Vitacos Cosmetics [
15,
16]. Moreover, the international cosmetics company L’Oreal patented a number of formulas for nanoemulsion cosmetics [
15,
16]. In the case of perfume products, nanoemulsions can be not only a medium for a fragrance composition, but they also increase chemical stability of the compounds in the composition (protection against oxidation) [
12,
13,
17,
18].
Alcohol-free perfumes based on micro- and nanoemulsions are already present in the patent literature [
19,
20,
21,
22], however, apart from additional solubilizers, such as polyols or paraffin hydrocarbons, the compositions of the perfumes in the above-mentioned patents have cationic or anionic surfactants, which can be an irritant to the skin. Nanoemulsions that serve as a matrix for fragrance substances used in cosmetics are also described in the literature [
18,
23,
24,
25,
26,
27,
28] these are primarily solutions for emulsification of a single lipophilic component that forms the oil phase of a nanoemulsion (e.g., D-limonene) [
18,
23,
24]. On the other hand, the fragrance compositions contain up to twenty compounds with various chemical structures (alcohols, phenols, aldehydes, esters, saturated, unsaturated, cyclic, and branched hydrocarbons), and as a result they are a difficult base for obtaining stable nanoemulsion systems.
Nanoemulsions turned out to be a great solution for solving problems related to the oxidation and low-bioavailability of fragrances and they could be applied as nano-encapsulated fragrance systems, in the perfume industry. Therefore, the most important aspect for further research would be to make the preparation of fragrance nanoemulsions practicable, at a pilot scale, so as to make them possible to be adopted in the industrial full-scale production.
The aim of the research was to develop stable oil-in-water (O/W) nanoemulsions that are compatible with selected fragrance compositions, without ethanol, polyols, and ionic surfactants, and to study the physicochemical, microbiological, and dermatological properties, as well as permanence of the fragrance of the obtained nano-perfumes. The nano-perfume systems were obtained with a low-energy method (Phase Inversion Composition; PIC), as well as with an ultrasound (US) high-energy method, taking into account the possibility of moving from the laboratory scale to an industrial scale.
2. Materials and Methods
2.1. The Properties of Raw Materials
In order to obtain dispersive systems with the droplet size of 20–500 nm of the inner phase, the research concerned the influence of the type and concentration of the oil phase (of a given fragrance composition) and of the type and concentration of the surfactant on the stability of nanoemulsion systems. The emulsifiers used in the study were supplied by the Croda company (Krakow, Poland) (
Table 1). Fragrance compositions were made by European Flavours & Fragrances PLC (Hertfordshire, UK). All tested fragrances are of GRAS (Generally Recognised As Safe) status, allergen free. Milli-Q® filtered water (Merck, Warsaw, Poland) was used as the aqueous phase of the nanoemulsions. The preservative used was Dermosoft 1388 (up to 1%), kindly supplied by Evonik Dr. Straetmans GmbH (Germany).
Nonionic surface active agents were used in the research. Those surfactants belong to the group of polyoxyethylated esters of glycerin and fatty acids, polyoxyethylated castor oil, polyglycerol and fatty acids esters, and alkyl polyglucosides. Surfactants of this type are known for their very good performance and dermatological properties. They show biocompatibility with the skin and they are used in cosmetics, such as solubilizers, humectants, dispersing agents, and emulsifiers, for the stabilization of O/W emulsions. In comparison to the ionic surfactants, both cationic and anionic, they are not susceptible to pH change and the addition of electrolytes. They can be used with other emulsifiers to increase the system stability [
13,
29,
30,
31].
2.2. The Method of Obtaining Nanoemulsions
2.2.1. Phase Inversion Composition Method (PIC)
To obtain perfumes in the form of a nanoemulsion, the low-energy method was used (Phase Inversion Composition—PIC). The nanoemulsions were obtained by a gradual addition of water to the mixture of a surfactant with the oil phase (the fragrance composition), at room temperature (25 °C), with constant stirring (IKA Vortex Genius 3 shaker).
2.2.2. Ultrasonic Homogenization Method (US)
The nano-perfumes in the nanoemulsion form were obtained with a high-energy method that requires ultrasonic homogenization with initial pre-emulsification. A specified amount of the oil phase (fragrance composition), surfactant, and demineralized water was dispersed at room temperature (25 °C), with a mechanical stirrer (IKA° RW 20 digital), 500 rpm, for 10 min. The obtained pre-emulsion underwent ultrasonic homogenization (probe-type sonicator UP200Ht, Hielscher Company, Teltow, Germany) with 15 W for at least 60 s.
2.3. Tests of the Physicochemical Properties of the Fragrance Compositions
In order to establish the properties of the fragrance compositions used in the research, their surface tension was measured with a tensiometer STA-1 by Sinterface (Berlin, Germany) with a du Noüy ring. Viscosity was determined with an R/S (Cone/Plate) rotational rheometer with cone/plate measuring elements (by Brookfield). Moreover, the value of logP was calculated (i.e., lipophilicity of the composition) on the basis of the qualitative and quantitative constituents of the used fragrance compositions provided in material data sheets. This was determined by calculating the lipophilicity (logP, P—Partition coefficient) of individual relevant constituents and the percentage composition of the fragrance [
32].
2.4. Tests of the Physicochemical Properties of the Nanoemulsion
In order to determine the physicochemical properties of the obtained systems, the following analytical methods and techniques were used. The size of the droplets of the inner phase was analyzed with a Zetasizer Nano ZS droplet analyzer (Malvern Instruments, Malvern, UK). The kinetic stability of the emulsion systems was monitored with the analysis of the droplet size of the inner phase of the nanoemulsion and the polydispersity index (PDI) over time. To determine the rheological properties of the obtained formulations, just like in the case of pure compositions, an R/S rotational rheometer was used with cone/plate measuring elements (cone C25-1), at room temperature (25 °C). Viscosity tests were conducted with a variable cutting rate, within the range of 1–500 rps. The surface tension of the emulsion was also measured with a tensiometer and a du Noüy ring, in the same way as in the case of the fragrance compositions. The pH value of the emulsion system was determined with a multi-functional measuring tool (Seven Multi by Mettler Toledo, Warsaw, Poland) which was equipped with an electrode for measuring pH. Density was established with a hydrostatic method, using an analytical balance (AS310X0, Radwag, Krakow, Poland), with a density measuring device.
2.5. The Test of the Microbiological Properties of the Nanoemulsion
The test of microbiological purity of the obtained stable products was conducted according to the following polish standards: PN-EN ISO 21149:2009, PN-EN ISO 162012:2011, PN-EN ISO 22718:2010, PN-EN ISO 21150:2010, and PN-EN ISO 18416:2009. The preservation tests were carried out according to the standard PN-EN ISO 11930:2012. The following sample strains were used: Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 11229, Candida albicans ATCC 10231, and Aspergillus brasilensis ATCC. With regard to every checked microorganism, the test involved the treatment of a cosmetic preparation with a standardized inoculum and then the measurement of the number change of the microorganisms, at given intervals, during a specified period and under a given temperature.
2.6. The Test of the Dermatological Properties of the Nanoemulsion
The dermatological test of the obtained products was conducted with a patch test on a group of twenty-five testers (15 women and 10 men, age 19–55), under a dermatologist’s supervision, in accordance with the applicable legal provisions and guidelines for dermatological research on humans. The tests were conducted in a specialist dermatological medical room. Patch tests were applied on the inner side of the forearm. The tested preparation was rubbed onto a 1 × 1 cm skin patch, the area with an applied cosmetic was covered with a 2 × 2 cm filter paper and foil attached with an adhesive. The dressing was taken off after 24 h and the result was assessed organoleptically. Further dermatological check was carried out after 3, 4, and 5 days, after application. The assessment was conducted according to the generally accepted scale for dermatological tests [
33].
2.7. The Test of Fragrance Permanence of the Nanoemulsion
The test of fragrance permanence was carried out in accordance with the standard BN-84/6148-02. Two strips of filter paper, 150 mm long and 15–20 mm broad, were dipped 20 mm in the samples; the first one was dipped in a tested sample, and the second one in a reference sample, and left at the temperature of 25 °C. The intensity of the fragrance was assessed organoleptically after 1, 24, and 48 h after dipping. The samples were assessed by comparing the intensity of the fragrance of the tested sample and the reference sample.
2.8. The Test of Chemical Stability of the Fragrance Composition (Oxidative Stress Test)
The oxidative stress test was carried out to demonstrate the chemical stability of the fragrances and protecting effect of nanoemulsions against peroxidation of the fragrance composition. The oxidation process induced by the oxygen from air and by UV, was determined by measuring the peroxide value of the fragrances, according to Wheeler DGF standard method C-VI 6a [
34]. The value was measured as the amount of iodine which was formed by the reaction of peroxides (formed in oil) with iodide ion. The sample was added to a mixture of glacial acetic acid and isooctane (60/40
v/
v) and then allowed to react with potassium iodide (0.5 cm
3 of saturated solution). The iodine released was determined by titration, using 0.01 N sodium thiosulfate solution. The titration end point was specified iodometrically. The peroxide value (POV) was calculated according to Equation (1):
where
a is the consumed volume of sodium thiosulfate solution,
b is consumed volume of sodium thiosulfate solution in the blank test,
M is the molarity of the sodium thiosulfate solution, and
Q is the quantity of the tested sample with accuracy ±0.1 mg. Each sample was assessed in triplicates (
n = 3).
2.9. Statistical Analysis
All data presented in the plots were presented as a mean of three different experiments ± SD. Differences between the calculated means of each individual group were determined by one-way ANOVA tests, using the statistical software Statistica Version 12 StatSoft Company., Cracow, Poland. A value of p < 0.05 was considered statistically significant.
4. Conclusions
As a result of the performed studies, stable oil-in-water (O/W) nanoemulsions were obtained, which were compatible with the six selected fragrance compositions, and they did not contain ethanol or any other solvent. These systems can be successfully applied as modern carriers of selected fragrance compositions, both with a low-energy method (PIC), at a laboratory scale, and with a high-energy method (US), at a production scale. The optimized nano-perfume recipes that were obtained with different methods, yielded the same physicochemical properties (stability, medium droplet size of the inner phase, polydispersity, viscosity, surface tension, pH, and density). The simple composition of the formulation is worth noting. The use of a nonionic surfactant that was gentle to the skin as an emulsifier, i.e., castor oil (Etocas 35) ethoxylated with 35 moles of ethylene oxide, allowed to obtain stable systems, without the need to use additional solubilizers, such as polyol, e.g. glycerin, an oil phase component, or an oil with low polarity (e.g., isohexadecane). Stable, transparent nano-perfumes were obtained with a fragrance composition concentration within 6–15% range [
50,
51]. These formulations have low viscosity and pH suitable for the skin. Moreover, the obtained results confirmed the protective role of nanoemulsions. The peroxide number measurement (POV) showed that the tested fragrance compositions had a high chemical stability. The results of the microbiological tests confirmed that the obtained products were free of microbiological contamination and were appropriately preserved. The dermatological test results confirmed the safety of the developed preparations.