1. Introduction
In recent years, eco-friendly products are at the forefront in the skin care industry [
1]. In line with the increased public awareness of environmental issues, many consumers are seeking eco-friendly skin care products in their purchases. Surveys reported that nowadays consumers are willing to pay more on eco-friendly products [
2,
3]. These heightened demands have driven various innovations along the skin care value chain, including formulation level to meet consumers’ expectations without compromising the quality and functionality of products. In order for the companies to stay ahead in the highly competitive skin care market, there is an urgent need for them to create eco-friendly products with greater product efficacy. Besides the traditional aesthetic effects, consumers nowadays are also expecting effective preventive and therapeutic effects with minimal side effects from the skin care products [
4,
5].
Delivery systems are used extensively in skin care products. They are incorporated in the skin care formulations to enable a controlled and targeted delivery of skin care actives, enhance penetration of skin care actives through the skin layers and provide protection to the skin care actives [
5,
6]. Microemulsions are among the advanced delivery technologies that have gained increasing attention from skin care formulators due to their good thermodynamic stability, superior solubilization power on lipophilic ingredients, appealing appearance (optically transparent) and ease of preparation [
7,
8]. These advantages have made microemulsions a promising alternative to overcome the major limitations of conventional emulsions. Microemulsions are clear isotropic mixtures of two immiscible liquids (such as oil and water) that are stabilized by surfactant or a mixture of surfactant with co-surfactant. Their droplet size is typically below 100 nm [
8,
9]. Whereas emulsions are coarse milky mixtures of two immiscible liquids that are stabilized by emulsifier, with droplet size in micron range [
10]. Distinct from conventional emulsions that are typically opaque in nature and have a very limited shelf life due to their tendency to break down over a short time via various destabilizing processes such as sedimentation, coalescence, flocculation and the Ostwald ripening [
11,
12]), microemulsions are thermodynamically stable and readily provide a transparent and elegance appearance to enhance consumer appeal. Furthermore, microemulsions are easily scalable by low input of energy and without the requirement of any sophisticated equipment [
8], making this technology platform simple and cost-effective from the manufacturing point of view.
Essential oils are volatile compounds extracted from different plant parts such as flowers, leaves, bark or roots through steam distillation or cold-pressing [
13]. It has been estimated that nearly three thousand essential oils have been identified and about three hundreds of them are of commercial importance [
14]. They are highly complex mixtures of chemical and bioactive constituents that not only provide pleasant aroma and flavour, but also offer various health benefits such as anti-inflammatory, antioxidant, anti-mutagenic, anticancer, and antimicrobial [
14,
15,
16]. These therapeutic properties have led to their long history of application in pharmaceutical, food and skin care. Essential oils have gained popularity as natural remedies in skin care products to treat various dermatological conditions and improve aesthetic benefits [
17,
18]. Moreover, they have also been explored as natural alternatives to artificial additives (e.g., preservatives and antioxidants) which are widely used in skin care formulations but found to be harmful to both consumers and environment [
19,
20].
Parabens are a family of esters derived from para-hydroxybenzoic acid that are used extensively as synthetic preservatives in skin care and cosmetic products such as shampoos, cleansers, moisturizers and makeup [
21]. It has been reported that nearly 99% of leave-on skin care and cosmetic products contain some forms of paraben [
22]. They are highly efficient in inhibiting microbial growth and hence are often used to prolong the shelf life of products and prevent the transfer of microorganism from the products onto the surface of the consumer’s skin (i.e., skin infections). Among the parabens, short chain parabens such as methylparaben and propylparaben are used most often in skin care and cosmetics products due to their lower toxicity. The shorter the alkyl chain, the lower the lipophilicity and toxicity of parabens [
23]. Nevertheless, they remain one of the most controversial ingredients in skin care products because of their potential endocrine disruption and increased risk of breast cancer [
24,
25,
26]. As parabens are structurally similar to estrogen, they may penetrate the skin and mimic the effects of physiological estrogen to promote the growth and proliferation of breast cancer cells [
27]. Besides parabens, BHT (butylated hydroxytoluene) is another synthetic ingredient that appears frequently in a wide range of skin care formulations (especially those are containing oils and fats) for stabilizing them against deterioration by free radicals [
28]. It is used for its antioxidant properties in reducing the oxidation reaction and intrinsic odour of the formulations, thereby to improve the shelf life and quality of products. The use of BHT in skin care formulations has been criticised and associated with adverse effects such as DNA repair failure, oxidative stress, and pulmonary toxicities [
29,
30,
31]. Although the safety risks of synthetic ingredients such as parabens and BHT remain controversial [
32,
33,
34], the current consumers are increasingly mindful about the ingredients used in skin care products in order to safeguard their own health. Consumers’ perception of the safety of synthetic ingredients has triggered skin care manufacturers to search for safer alternatives to replace or reduce these synthetic ingredients in the skin care products. Multifunctional ingredients, which are added to the skin care formulations for a well-defined function while can concurrently contribute to other beneficial effects (e.g., antioxidant or antimicrobial activity), have emerged as the popular ingredients in formulating gentler and safer skin care products.
The aim of this work was to explore and investigate the feasibility of developing eco-friendly skin care formulations using microemulsions of essential oil. While microemulsions of essential oil act as the novel delivery systems in an attempt at improving the release (membrane permeation) and stability of the skin active, essential oils were innovatively used as the safer natural alternatives to synthetic antioxidants and preservatives in the skin care formulations. Naringin (chemical structure shown in
Figure 1) was chosen as the skin care active in this work because of its excellent pharmacological actions such as anti-inflammatory, antioxidant and antiviral characteristics that are highly favourable in skin care application [
35,
36,
37]. However, formulating plant-based skin care active (e.g., naringin) could be challenging due to its stability issues [
38]. Moreover, being a BSC class II drug, the skin care application of naringin is impeded by its poor water solubility [
37]. Nagase’s newly developed Glucosyl Naringin (combination of glucose with naringin) presents one of the efforts to improve water solubility of naringin for skin care application [
39]. In this work, microemulsions were exploited to enhance the release rate of poorly water-soluble naringin. To the best of our knowledge, no naringin-loaded microemulsion has been reported in the literature. The role of microemulsions of essential oil in the skin care formulations was envisioned to be 4-fold: (1) to improve the release (membrane permeation) of skin active, (2) to improve stability of skin active, (3) as a natural alternative to synthetic antioxidant, and (4) as a self-preserving system.
2. Materials and Methods
2.1. Materials
Peppermint oil (PMO), lavender oil (LVO) and eucalyptus oil (EUO) were purchased from Euro Chem-Pharma Sdn. Bhd. (Johor Bahru, Malaysia). Naringin, Tween® 80 (polysorbate 80), diethylene glycol methyl ether (DGME), orthophosphoric acid, 2,2-diphenyl-1-picrylhydrazyl (DPPD), butylated hydroxytoluene (BHT), methylene blue and Sudan III were supplied from Sigma Chemical Co. (St. Louis, MO, USA). SimulgelTM NS (INCI name: hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer and squalane and polysorbate 60) was a gift sample from Seppic (Courbevoie, France). HPLC grade acetonitrile (ACN) and absolute ethanol (EtOH) were obtained by Merck (Darmstadt, Germany). Ultra-pure water (Milli-Q® Gradient A10®, Millipore, Molsheim, France) was used in the experiments.
2.2. Construction of Pseudo-Ternary Phase Diagrams
Pseudo-ternary phase diagrams were constructed using the water titration method to identify the microemulsion region of each oil-surfactant/co-surfactant-water system in this work. Firstly, the surfactant phase (surfactant/co-surfactant mixture, Smix) was prepared by mixing surfactant (Tween® 80) and co-surfactant (EtOH or DGME) at various weight ratio such as 1:1, 1:2 and 2:1. The accurately weighed oil phase (PMO, LVO or EUO) was then added into each surfactant phase at weight ratios of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1. Each mixture of oil and surfactant phase was homogenously mixed with a vortex mixer (Vortexer, Heathrow Scientific, Vernon Hills, IL, USA), followed by titration with multiple 10 µL aliquot of ultrapure water (water phase) until turbidity was visually observed. The amount of each component (oil, surfactant/co-surfactant, and water) to form clear microemulsions was calculated as weight percent and plotted on a triangular graph as the pseudo-ternary phase diagram.
2.3. Preparation of Naringin-Loaded Microemulsions and Microemulsion-Gel Formulations
Six microemulsions (MEs) at different component ratios were selected from the microemulsion regions in the pseudo-ternary phase diagrams and the naringin-loaded microemulsions (NMEs) were prepared (
Table 1). To prepare naringin-loaded microemulsions, naringin was first dissolved in the oil phase. Next, the oil phase containing naringin was mixed with mixture of surfactant/co-surfactant until homogenous. The appropriate amount of ultra-pure water was then added dropwise into the mixture of oil-naringin-surfactant/co-surfactant under constant magnetic stirring.
For the preparation of microemulsion-gel (MEG) formulations, Simulgel
TM NS was used as the thickener as it is an environmentally friendly produced thickener with good stability and texturizing properties [
40]. Between 2–3% (
w/w) of the ultra-pure water in the microemulsions was replaced by Simulgel
TM NS (
Table 1). For the preparation of unformulated naringin plain gel (as reference gel), naringin was dissolved in ethanolic solution (ethanol: water, 10:90
v/v). 2% (
w/w) of the ultra-pure water in naringin ethanolic solution was substituted with Simulgel
TM NS. Simulgel
TM NS was added slowly to the microemulsions (for preparing microemulsion-gel formulations) or ethanolic solution (for preparing naringin plain gel) while stirring with an overhead stirrer (Hei-TORQUE Core, Heidolph Instruments GmbH & CO. KG, Schwabach, Germany) at 150 rpm for 20 min. The final concentration of naringin in both microemulsion, microemulsion-gel formulations and plain gel was set to be 1% (
w/w).
2.4. Characterization of Naringin-Loaded Microemulsions and Microemulsion-Gel Formulations
2.4.1. Particle Size and Polydispersity Index
The particle size (z-average diameter) and polydispersity index (PdI) of the microemulsions were measured by dynamic light scattering technique (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK) at 25 °C.
2.4.2. pH Measurements
The pH of naringin-loaded microemulsions and microemulsion-gel formulations were measured using a benchtop pH meter (SevenExcellence S470, Mettler-Toledo AG, Schwerzenbach, Switzerland) at 25 °C.
2.4.3. Viscosity Measurement
The viscosity of naringin-loaded microemulsions and microemulsion-gel formulations were determined using a rotational viscometer (model DV2T, Brookfield, Stoughton, MA, USA) with spindle RV-03 at speed 150 rpm and spindle RV-05 at speed 20 rpm, respectively, at 25 °C.
2.4.4. Determination of Types of Microemulsion (via Dye Solubility and Conductivity Test)
The type of microemulsion (i.e., O/W or W/O microemulsion) was qualitatively evaluated via dye solubility test using methylene blue (water-soluble dye) and Sudan III (oil-soluble dye). An equal amount of each dye sample was separately added to microemulsions and the diffusion rate of each dye in the microemulsions was observed visually. The diffusion rate of methylene blue is observed faster than Sudan III in O/W microemulsions and slower than Sudan III in W/O microemulsions [
41].
Electrical conductivity of microemulsions was measured using a benchtop conductivity meter (SevenExcellence S470, Mettler-Toledo AG, Schwerzenbach, Switzerland) fitted with an InLab® 741 conductivity probe with a cell constant of 0.105 cm−1 (Mettler-Toledo AG, Schwerzenbach, Switzerland) at 25 °C.
2.5. High Performance Liquid Chromatography (HPLC) Assay of Naringin
The assay of naringin was conducted by reversed phase HPLC (1100 series, Agilent Technologies, Santa Clara, CA, USA) equipped with a ZORBAX Eclipse Plus C18 column (4.6 mm × 150 mm, 3.5 μm) (Agilent Technologies, Santa Clara, CA, USA). A modified HPLC method [
42] with a mobile phase consisting of a mixture of ACN (70:30
v/v) and 0.1% phosphoric acid was used. The flow rate, retention time and detection wavelength of quercetin were 1 mL/min, 2.5 min and 289 nm, respectively.
2.6. Stability
Naringin-loaded microemulsion and microemulsion-gel formulations were stored in 8-mL stoppered glass vials with PTFE/white silicone septa black solid cap (Newton 101 Pte. Ltd., Singapore) and equilibrated for one month at refrigerated conditions (4 °C/65% RH), ambient conditions (25 °C/60% RH) and accelerated conditions (40 °C/75% RH) (Binder KBF 115 RH Chamber, BINDER GmbH, Tuttlingen, Germany). After the storage period, the chemical stabilities of naringin in the microemulsion and microemulsion-gel formulations were analysed by HPLC and physical stabilities were assessed by visual inspection of samples for any signs of phase separation, precipitation or colour change. The stabilities of microemulsions were also accessed by measuring the particle size (z-average diameter) and polydispersity index (PdI) of microemulsion as described in
Section 2.4.1.
2.7. In Vitro Release (Membrane Permeation) of Naringin
In vitro release (membrane permeation) of naringin from the microemulsion-gel formulations were conducted on static vertical Franz diffusion cells equipped with 20-mL receptor compartments, clamps, and stirrer bars on a six-cell drive system (V6B, PermeGear Inc., Hellertown, PA, USA). Each receptor compartment was filled with a receptor medium consisting of 80% (v/v) phosphate buffer saline (PBS, pH 7.4) and 20% (v/v) methanol to maintain sink condition. The receptor medium was maintained at 32 ± 0.5 °C by a circulating water-bath (HAAKE S5P and SC100, Thermo Scientific, Waltham, MA, USA). When the system was equilibrated and stable, 200 ± 2 mg of microemulsion-gel formulation was evenly spread on the synthetic polyethersulfone membrane (Supor® 450, PALL Life Sciences, Ann Arbor, MI, USA) and sealed with a clamp over the membrane. 0.7 mL sample aliquots were withdrawn from the receptor compartment through the sampling port at regular time intervals (0.5, 1, 2, 3, 4, 5 and 6 h) and analysed for their concentrations using the previously described HPLC method.
2.8. In Vitro Antioxidant Activity
In vitro antioxidant activity of active-free microemulsions of essential oil (MEs) was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [
43] with some modifications. Briefly, 1 mL of active-free microemulsion of essential oil was mixed with 1 mL of 0.15 mM DPPH solution in ethanol. The mixture was kept in a dark room for 20 min at room temperature before the absorbance of the mixture was measured using a UV–VIS spectrophotometer (Cary 60, Agilent Technologies, Santa Clara, CA, USA) at 517 nm. The antioxidant activity (represented by percentage of inhibition) of sample was calculated as follows:
where A
o was the absorbance of control (mixture of equal volume of DPPH solution and EtOH) and A
s was the absorbance of sample (mixture of equal volume of DPPH solution and test sample). BHT was used as the standard reference.
2.9. In Vitro Antimicrobial Activity
In vitro antimicrobial activity of active-free microemulsions of essential oil (MEs) were evaluated using the disc diffusion method [
44] against
E. coli and
S. epidermidis. The diluted bacterial inoculums (~10
6 CFU mL
−1) were spread on trypticase soy agar (TSA) using sterile cotton swabs. Then, the sterile discs (6 mm diameter, Whatman No. 1, Whatman Ltd., Maidstobe, UK) were placed on surface of inoculated agar plates and impregnated with 20 µL of active-free microemulsions of essential oil and control. All the active-free microemulsions of essential oil and control were sterilized using 0.22 µm PTFE membrane (Sterlitech Corporation, Auburn, WA, USA) filtration method. Moreover, 0.25% (
w/v) aqueous solution of methyl paraben was used as positive control. The agar plates were incubated in an incubator (LE-80D, Yihder Co., Ltd., Xinbei, Taiwan) at 37 °C for 24 h and the diameters zone of inhibitions were measured.
4. Conclusions
This work demonstrated the feasibility of developing eco-friendly skin care formulations using microemulsions of essential oil. Six microemulsions of essential oil containing peppermint oil, lavender oil or eucalyptus oil, surfactant Tween® 80 and co-surfactant EtOH or DGME (i.e., ME-1, ME-2, ME-3, ME-4, ME-5 and ME-6) have been successfully developed and optimised via pseudo-ternary phase diagram. The essential oils used to formulate microemulsions were multifunctional ingredients that not only served as the oil phase of microemulsion but also exhibited antioxidant and antimicrobial properties. All of the six active-free microemulsions of essential oil showed an antioxidant activity equivalent to at least 25 µg/mL of synthetic antioxidant BHT, with the inhibition level of ME-3 and ME-4 as high as ~60% (approximately equivalent to antioxidant activity of 100 µg/mL BHT). Favourably, E. coli and S. epidermis strains were found to be more susceptible to all of the sixactive-free microemulsions of essential oil than 0.25% (w/w) synthetic preservative methyl paraben. These findings suggest the potential of microemulsions of essential oil as a safer alternative to synthetic antioxidant and as a self-preserving system. When loaded with 1% (w/w) of poorly water-soluble naringin and formulated into gel form, these naringin-loaded microemulsion-gel formulations (NMEG-1, NMEG-2, NMEG-3, NMEG-4, NMEG-5 and NMEG-6) demonstrated a significantly higher release of naringin and better stability than the unformulated naringin plain gel (microemulsion-free naringin gel). Therefore, a 4-fold benefit of microemulsion of essential of oil has been demonstrated in the developed naringin-loaded microemulsion-gel formulations: (1) improved release (membrane permeation) of naringin, (2) improved stability of naringin, (3) as a natural alternative to synthetic antioxidant such as BHT and (4) a self-preserving system. The application of microemulsions of essential oil in skin care and cosmetic formulations has indeed presented the opportunities for the formulators to develop eco-friendly skin care products while seeking more improvements in the product efficacy.