PHOTOPROTECTIVE EFFICACY OF ROBUSTA GREEN COFFEE (Coffea canephora) EXTRACT AND LINSEED (Linum usitatissimum) OIL-BASED NANOEMULSIONS AGAINST UVB-INDUCED DAMAGE TO THE SKIN

This study adopted a preclinical model to develop and evaluate the photoprotective efficacy of nanoemulsions (NEs) containing Robusta green coffee extract (GCE) and linseed oil (LO) against UVB-induced skin damage. The GCE, rich in polyphenols and caffeine, demonstrated antioxidant activity in vitro. The LO met quality standards based on physicochemical analyses (peroxide, acid, and saponification values), confirming its suitability for formulation. Using Chremophor^® RH 40 and Span^® 80 as surfactants yielded stable and nanometric NEs, since both the control with only LO (NE-C) and the NEs loaded with GCE at concentrations of 0.25% (NE-0.25), 0.5% (NE-0.5), and 1.0% (NE-1.0) showed D50 < 90 nm, PDI ≤ 0.2, and ZP < -26.0 mV. Morphological studies confirmed a spherical shape and uniform droplet distribution, with no aggregation. Moreover, all NEs presented a pH of around 5, compatible with human skin. The NEs were topically applied to the dorsal skin of hairless mice before and after UVB exposure. Besides being well tolerated following an acute irritation test, NE-0.25 markedly reduced neutrophil migration (49%) while recovering the skin antioxidant capacity (catalase activity and reduced glutathione levels) to near-baseline values within 12 h of UVB exposure, thus highlighting its potential as a skin anti-aging strategy.

Keywords:
Antioxidants; Coffea; Flax; Functional Food; Nanotechnology; Skin Aging; Ultraviolet Rays.

GRAPHICAL ABSTRACT

INTRODUCTION

Excessive exposure to solar radiation poses significant challenges to skin health, since it has been closely related to adverse effects on skin integrity and function (Bowden, 2004; Afaq, Adhami, Mukhtar, 2005; Bosch et al., 2015; Khan et al., 2020), thus leading to several dermatological disorders, e.g., inflammation, hyperplasia, erythema, edema, sunburn, immunosuppression, premature skin aging, and photocarcinogenesis (Bowden, 2004; Afaq, Adhami, Mukhtar, 2005; Ciążyńska et al., 2021).

Among the different wavelengths of UV radiation, UVB (280-315 nm) has the greatest potential to cause cellular damage and mutations in human skin. The primary consequence of UVB radiation is a direct interaction with the DNA of skin cells, which can result in the development of malignancies (Ansary et al., 2021; Ciążyńska et al., 2021). Additionally, UVB radiation induces indirect damage by generating reactive oxygen species (ROS), overwhelming the skin’s natural antioxidant defense mechanisms and leading to oxidative stress. When oxidative stress becomes excessive, it triggers an inflammatory response and increases the risk of developing skin cancer (Saito et al., 2018; Ansary et al., 2021; Ciążyńska et al., 2021).

Various prophylactic and therapeutic strategies have been implemented to attenuate the adverse effects of UVB radiation on the skin, among which sunscreens are the most popular intervention. However, concerns have been raised regarding the efficacy and safety of the synthetic UV filters used in their formulations. These compounds can undergo photodegradation, generating toxic byproducts that may interact with cells, damaging DNA and compromising the intended photoprotective action of sunscreens (Kockler et al., 2012).

Sunscreens are often marketed as conventional emulsions and associated with several patient complaints, e.g., an unpleasant smell, difficulty spreading, a greasy or sticky feel, a heavy or thick texture, a white cast, irritations, breakouts, and acne (Addor et al., 2022; Portilho et al., 2022). Scientific evidence indicates that these substances may disrupt endocrine function, interfering with the normal physiological processes in the human body (Lorigo, Mariana, Cairrao, 2018). Moreover, there is growing concern about the toxicological impacts of UV filters on marine organisms (Jesus et al., 2022).

Given the well-established relationship between increased oxidative stress and the harmful effects of UV radiation, in addition to reports raising concerns about sunscreen use, supplementing the skin with plant-derived natural antioxidants emerges as a feasible alternative for photoprotection (Nichols, Katiyar, 2010; Saewan, Jimtaisong, 2015; Petruk et al., 2018).

Indeed, oral and topical administration of herbal extracts and functional foods has been suggested for preventing UV-induced sunburn response, DNA damage, inflammation, and skin aging (Hernandez et al., 2021; Ghazi, 2022; Fernandes et al., 2023). The photoprotective activity of these phytocomplexes has been attributed to copious phytochemicals, with diverse chemical structures and outstanding antioxidant capacity. Of note, our research group has extensively investigated the potential of herb-derived antioxidants in protecting the skin against damage caused by UVB irradiation (Campanini et al., 2013; Martinez et al., 2020; Takayama et al., 2022).

Coffee is a complex beverage comprising over a thousand identified compounds, that contribute to its aroma, enjoyable taste, and potential health benefits. These bioactive components act synergistically to provide antioxidant, anti-inflammatory, antibacterial, hepatoprotective, neuroprotective, and anticancer properties (Nigra, Teodoro, Gil, 2021). Notably, phenolic compounds, especially chlorogenic acids (CGAs), and caffeine found in coffee have demonstrated efficacy in mitigating UV radiation-induced damage and acting as potent scavengers of ROS (Kerzendorfer, O’Driscoll, 2009; León-Carmona, Galano, 2011; Herman, Herman, 2012).

The Coffea canephora Pierre ex A. Froehner (Robusta coffee) and Coffea arabica L. (Arabica coffee) species hold significant economic value worldwide (Vignoli et al., 2014). A study reported that Robusta coffee exhibits higher antioxidant capacity than Arabica coffee (Daglia et al., 2004), making Robusta coffee extracts the target of photoprotection investigations.

Likewise, LO is an edible plant derivative with remarkable therapeutic effects. It is recognized as a natural reservoir of omega-3 PUFA, mainly α-linolenic acid (ALA) (Buckner et al., 2019; Shadyro et al., 2022). The capacity of LO to mitigate inflammatory diseases and suppress the cancerous tumor growth has been associated with ALA (Michotte et al., 2011; Buckner et al., 2019), which could also contribute to its likely anti-inflammatory capacity in the skin (Buckner et al., 2019; Kouamé et al., 2021). In addition, LO contains appreciable amounts of linoleic acid, an omega-6 PUFA (Buckner et al., 2019), dietary fiber, protein, and phenolic compounds, predominantly lignans, contributing to its antioxidant capacity (Sargi et al., 2013).

Vegetable oils, such as LO, have been used as a core component in skincare emulsions, mainly because of their safety, biocompatibility, and complex fatty acid composition, contributing to skin hydration, UV radiation protection, and inflammation control (Bajerski et al., 2016; Poljšak, Kreft, Kočevar Glavač, 2020; Fagionato Masiero et al., 2021; Kunik et al., 2022). However, the high content of PUFA in LO makes it prone to lipid degradation, thus leading to low chemical stability (Michotte et al., 2011; Pereira et al., 2016). Therefore, using this active ingredient in the development of topical drug delivery systems represents a complex task.

Developing o/w NEs, i.e., a non-conventional liquid/ liquid colloidal dispersion characterized by oily globule sizing from 20 to 200 nm (Jaiswal, Dudhe, Sharma, 2015; Ali et al., 2017), is a feasible strategy to circumvent these issues, because these NEs act as carriers for lipophilic actives in the inner or dispersed phase (Jaiswal, Dudhe, Sharma, 2015) and effectively safeguard them against degradation (Sivakumar, Tang, Tan, 2014).

NEs can be prepared using simple, prompt, and affordable methods (Pereira et al., 2016; Azmi et al., 2019; Javadi, Kazemi, Halabian, 2021) that require low concentrations of surfactants, thus minimizing the potential for skin irritation (Bouchemal et al., 2004; Jaiswal, Dudhe, Sharma, 2015). Their smaller droplet size enhances physicochemical stability, penetration, and the retention of active ingredients in the skin, compared to conventional emulsions such as creams and lotions (McClements, Rao, 2011; Zhang, McClements, 2018; Krisanti, Kirana, Mulia, 2021). Furthermore, the attractive sensory characteristics of NE, such as their transparency, fluidity, and lightweight nature, contribute to their widespread acceptance for topical product application.

Despite the aforementioned attractive functional properties of Robusta coffee and LO, their combined effects in topical formulations for preventing and treating skin aging remain underexplored. Herein, we shed light on the development of NEs combining GCE and LO. These NEs concentrated biocompatibility and suitable organoleptic features, stability, and physicochemical properties, as well as providing effective photoprotection following topical administration against UVB-induced oxidative skin injury in hairless mice.

MATERIAL AND METHODS

Chemicals, reagents, and samples

Quercetin (99% purity, CAS 117-39-5) was obtained from Acros Organics (Geel, Belgium). Folin Ciocalteau reagent (CAS 9005-15-6) and gallic acid (CAS 149-91-7) were purchased from Fluka Chemical Co. (São Paulo, Brazil). DPPH (CAS 1898-66-4), ABTS (CAS 30931-67-0), TPTZ (CAS 3682-31-3), luminol (CAS 521-31-3), horseradish peroxidase (HRP, CAS 9003-99-0), DTNB (CAS 69-78-3), 5-caffeoylquinic acid (5-CQA) (purity ≥ 95%, CAS 327-97-9), caffeine (purity ≥ 98%, CAS 58-08-2), o-dianisidine dihydrochloride (CAS 119-26-6), hexadecyltrimethylammonium bromide (HTAB, CAS 57-09-0), Trolox (CAS 53188-07-1), PEG-40 hydrogenated castor oil (Cremophor^® RH 40, CAS 61791-12-6), and sorbitan monooleate (Span^® 80, CAS 1338-43-8) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid (CAS 64-19-7) was obtained from Merck (Darmstadt, Germany), and acetonitrile (CAS 75-05-8) was purchased from J.T. Baker (Phillipsburg, NJ, USA).

The aqueous freeze-dried GCE obtained from C. canephora species (lot 17, sample 101012965) was provided by Cia Iguaçu de Café Solúvel (Cornélio Procópio, PR, Brazil). The LO, from Pazze Indústria de Alimentos (Panambi, RS, Brazil), was purchased from a local store. Ultrapure water (conductivity of 0.1 μS/ cm) was obtained through a Milli-Q^® system (Millipore S.A., Molsheim, France). All other reagents used were of pharmaceutical grade.

Analytical standardization of GCE

The simultaneous quantification of caffeine and 5-CQA content was performed by adapting the HPLC-UV method, as previously described (Corso, Vignoli, Benassi, 2016). A chromatographic system from Thermo Fisher Scientific (Waltham, MA, USA) was used, equipped with a quaternary pump system, an autosampler, coupled to a diode array detector Libra S22 (Biochrom, Cambridge, UK), and controlled by Chromeleon^TM 7.0 software (Thermo Fisher Scientific, Waltham, MA, USA).

The GCE was diluted in acetic acid (5%) aqueous solution (0.1 mg/mL), and the samples were filtered through a membrane, 0.22 µm pore size (Millipore, São Paulo, Brazil). The injection volume was 25 µL and chromatographic separations were carried out using a Spherisorb^® ODS-1 column (150 mm x 4.6 mm, 3 µm) from Waters Co. (Milford, MA, USA). The mobile phase comprised acetic acid (5%) aqueous solution (A) and acetonitrile (B) and the compounds were eluted at a 0.6 mL/min flow rate using the following gradient: 0-1 min (5% B) and 1-12 min (13% B). Detection was performed at 272 nm for caffeine and 320 nm for 5-CQA.

Quantification was performed by external standardization, plotting analytical curves in the range of 3.0 to 20.0 µg/mL for caffeine (R² > 0.99, p < 0.001) and 0.5 to 30.0 µg/mL for 5-CQA (R² > 0.99, p < 0.001). 5-CQA was used as the standard for estimating the total content of CGAs by summing the areas of the detected compounds. The experiments were performed in triplicate and the results were expressed in percentages (m/m, dry basis), as mean ± SD.

The Total Phenolic Content (TPC) in the GCE was determined using a spectrophotometric method, based on the complexation with the Folin-Ciocalteau reagent (Georgetti et al., 2006). Gallic acid was used as the analytical standard to plot an analytical curve (4.0 - 22.0 µg/mL, R² = 0.99) and the results were expressed in mg/g (dry basis). In turn, the Total Flavonoid Content (TFC) was evaluated using the spectrophotometric method, based on the complexation with AlCl₃ (Georgetti et al., 2007). Quercetin was used as the analytical standard to plot an analytical curve (8.0 - 28.0 µg/mL, R² = 0.99) and the results were expressed in mg/g (dry basis). Both analyses were carried out in triplicate using an Evolution 60 UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Assessing the in vitro Antioxidant Activity of GCE

The ferric-reducing antioxidant power of GCE (100 μg/mL) was determined at 595 nm (Sánchez-González, Jiménez-Escrig, Saura-Calixto, 2005). An analytical curve of Trolox (4.0 - 20.0 μM) was used to calculate the result in μM of Trolox equivalent per μg/ mL of extract. The DPPH and ABTS assays were used to measure the ability of GCE to scavenge these free radicals through the decrease in absorbance (Georgetti et al., 2006; Casagrande et al., 2007).

The chemiluminescence assay using the H₂O₂/ luminol/HRP system was employed to evaluate the capacity of GCE (0.01-5.0 μg/L) to inhibit the luminescence emitted by luminol in the presence of free radicals formed by the action of peroxidase on H₂O₂ (Georgetti et al., 2003). All measurements were performed in triplicate, and the results were reported as the concentration at which the CGE exerts half of its maximal inhibitory effect (IC₅₀, µg/mL). The IC₅₀ was estimated using a hyperbolic curve, and the results were expressed as the mean ± SD.

Quality assessment of LO

The identity and quality of LO were determined using official methods and recommended practices. The acidity index was evaluated following the 325/IV method (Instituto Adolfo Lutz, 2008). The saponification and peroxide indices were determined following the Anvisa methodology (Brasil, 2024). The relative density was assessed according to Anvisa using a glass pycnometer (Brasil, 2024), and the oxidative stability was tested using the Rancimat method (American Oil Chemists’ Society - AOCS, 2009). All tests were performed in triplicate, and results were expressed as mean ± SD.

Preparation and Characterization of NE

The o/w NEs containing LO but not GCE (control, NE-C) and the o/w NEs comprising various combinations of GCE with LO were prepared using a low-energy EPI method described elsewhere (Pereira et al., 2016). The mixture of surfactants (Span^® 80, HLB of 4.3; and Chremophor^® RH 40, HLB of 13.0) equivalent to 5% (m/m) of the final NE weight was dispersed in LO (5% m/m). The proportions and amounts of Span^® 80 and Chremophor^® RH 40 in the mixture were theoretically calculated based on the HLB required by the LO. This was previously evaluated as HLB = 10.0 (data not shown).

The water and oily phases were separately heated to 75 ± 2°C. The water phase was then slowly added to the oily phase under constant stirring at 600 rpm. To minimize any potential thermal degradation, immediately after emulsification the NEs were rapidly cooled to room temperature (25 ± 2 °C) using an ice bath. To prepare NEs enriched with the extract, GCE was added at different concentrations: 0.25 % (m/m) for NE-0.25, 0.5 % (m/m) for NE-0.5, and 1.0 % (m/m) for NE-1.0. In readiness, the dried CGE was diluted in 3.0 mL of ultrapure water and incorporated into the NE while stirring at 600 rpm for 3 minutes. Afterward, 0.5% (m/m) of a preservative solution comprising phenoxyethanol and parabens was added. The NE-C was prepared following the same procedure as the GCE-loaded NE. The only difference was that GCE was not incorporated into the formulation.

Various NEs were evaluated regarding macroscopic features, pH, conductivity, rheology, droplet size, polydispersity index (PDI), zeta potential (ZP), particle morphology, TPC, in vitro antioxidant capacity, and preliminary stability. Further methodological details are provided in the Supplementary Material. All tests were performed in triplicate and results were expressed as mean ± SD.

In vivo Studies

Animals

Male adult hairless mice (HRS/J), weighing approximately 30 g, were obtained from the University Hospital of Londrina State University. The mice were housed in cages under controlled temperature (23 ± 2°C), with free access to water and food, and maintained on a 12 h light and 12 h dark cycle. All experimental procedures were performed according to the guidelines of the Animal Ethics Committee (O. Circ. CEEA No. 19/2020 on February 27th, 2020, process No. 110197.2020).

Acute Irritation Test

The animals were divided into four groups, each containing three animals, i.e., i) NE-C, ii) NE-0.25, iii) NE-0.5, and iv) NE-1.0. A 300 μL aliquot of the various NEs was applied to the backs of the animals, previously washed, and the areas were observed at 24, 48, and 72 h to detect any signs or symptoms of inflammation or other abnormal changes in the skin (Balata et al., 2018; Ghosh, Mondal, Ramakrishna, 2019).

UVB-induced Oxidative Damage

The mice were randomly divided into six groups of six animals each, as follows: a) non-UVB-induced control, b) UVB-induced control, c) UVB-induced and treated with NE-C, d) UVB-induced and treated with NE-0.25, e) UVB-induced and treated with NE-0.5, and f) UVB-induced and treated with NE-1.0. The animals received a topical application of 300 μL of the NE on the dorsal area, administered at three distinct time points: 1) 1 hour before radiation, 2) immediately before radiation (5 minutes prior), and 3) immediately after radiation. Therefore, the total volume administered per animal was 900 μL. Based on this total dose, the amounts of phytochemicals administered per animal were as follows: 274.5 μg of CGA and 81.0 μg of caffeine for NE-0.25; 549.0 μg of CGA and 162.0 μg of caffeine for NE-0.5; and 1098.0 μg of CGA and 324.0 μg of caffeine for NE-1.0.

This administration schedule was performed to maximize the protective effect of the NE against UVB-induced oxidative damage, ensuring the presence of the antioxidant components throughout the exposure and early inflammatory response. This protocol was adapted based on previously published studies that used multiple topical applications to simulate preventive and immediate post-exposure interventions in UVB-induced skin injury models (Casagrande et al., 2006; Campanini et al., 2013).

During the radiation session, all groups were exposed simultaneously to a fluorescent UVB lamp (PHILIPS TL/12 40 W RS, Medical-Netherlands) to induce oxidative stress and acute inflammatory processes. The UVB dose used was 4.14 J/cm² (Casagrande et al., 2006; Campanini et al., 2013). The lamp emitted continuous irradiation at 270 to 400 nm, with peak emission at 313 nm. After 12 h of UVB exposure, the mice were euthanized with 5% isoflurane, and skin samples were collected. Leukocyte recruitment was indirectly quantified by MPO enzyme activity assay (Campanini et al., 2013) and the antioxidant capacity was evaluated using FRAP and ABTS methods. Each test was conducted twice, with 6 mice per group in each experiment. Results were presented as the mean ± SEM.

To measure MPO activity, the tissue sample was homogenized with 400 μL of a solution containing 0.05 M phosphate buffer (pH 6.0) and 0.5% HTAB. The homogenate was centrifuged at 16,400 x g for 2 minutes at 4°C. For the reaction, 200 μL of a solution containing 0.0167 mg/mL o-dianisidine in 0.05 M phosphate buffer (pH 6.0) and 0.015% hydrogen peroxide was mixed with a 30 μL aliquot of the supernatant. The absorbance of the mixture was determined at 450 nm and compared to a neutrophil standard curve. The results were expressed as neutrophils per mg of skin (Casagrande et al., 2006; Melo et al., 2021).

The skin sample was homogenized in 400 μL of KCl (1.15%) using a Tissue-Tearor (Biospec, Rio de Janeiro, RJ, Brazil) and centrifuged (1000 x g, 10 min, 4 °C). A 30 μL aliquot of the supernatant was reacted with the FRAP reagent, and the absorbance was measured at 595 nm. In parallel, a 7 μL aliquot of the supernatant was reacted with ABTS solution, and the absorbance was measured after 6 min at 730 nm. The results were compared to a Trolox curve (0.01-20 nmol) and expressed as nmol Trolox equivalent per mg of skin (Katalinic et al., 2005; Campanini et al., 2014; Martinez et al., 2015).

Statistical Analysis

The Least Squares Method allowed regression and correlation analysis of analytical data. The IC₅₀ was estimated using a hyperbolic curve, and the results are expressed as the mean ± SD. Pairwise comparisons were made using Tukey’s T-test. For the in vitro and in vivo results, statistical analysis of the experimental data of three or more samples was performed using one-way ANOVA followed by Tukey’s multiple comparison test, considering a significance level of p < 0.05 at a 95% confidence interval. Data were analyzed using GraphPad Prism^® 7 software (Boston, MA, USA).

RESULTS AND DISCUSSION

Phytochemicals and in vitro antioxidant activity of GCE

The GCE contained 12.2% (m/m) of CGA and 3.6% (m/m) of caffeine, as well as TPC of 155.3 mg/g (gallic acid equivalent) and TFC of 2.8 mg/g (quercetin equivalent). The chromatograms obtained in the analytical standardization are presented in Figure S1 of the Supplementary Material.

In the FRAP assay, the reducing power of GCE was determined to be 0.93 μM Trolox equivalent per μg/mL of extract. The result was calculated using the Trolox standard curve (y = 0.039x + 0.008, R²> 0.99). As presented in Figure 1, GCE showed a concentration-dependent scavenging effect on DPPH and ABTS radicals, with IC₅₀ values of 8.6 μg/mL (Figure 1a) and 3.8 μg/mL (Figure 1b), respectively. GCE also exhibited a concentration-response relationship in the chemiluminescence assay using the H₂O₂/luminol/HRP system, with an IC₅₀ of 0.3 μg/mL (Figure 1c).

FIGURE 1
In vitro antioxidant efficacy of Robusta green coffee extract using different methods: (A) hydrogen-donating ability to DPPH, (B) electron-donating ability to ABTS, and (C) scavenging activity against free radicals generated by H₂O₂/luminol/HRP system.

The results endorse the diverse mechanisms of the antioxidant action of phytochemicals contained in Robusta coffee. The antioxidant activity of standardized GCE was more pronounced in the ABTS assay than in the DPPH assay. This can be understood through the differences in their principles, mechanisms, and applications. On the one hand, the DPPH method is more specific to hydrophobic antioxidants, while on the other hand, ABTS can be used to assess both hydrophilic and lipophilic compounds (Munteanu, Apetrei, 2021). Therefore, the higher antioxidant capacity observed in the ABTS assay can be attributed to its greater sensitivity in detecting hydrophilic antioxidants contained in the GCE.

Quality assessment of LO

LO exhibited a golden color, characteristic odor, and relative density of 2.21 g/mL. The acid value was 2.5% oleic acid which complies with the acceptable limit specified by AOCS (i.e., less than 3%) (Firestone, 2006). The peroxide index was 4.9 mEq/Kg, below the maximum limit of 15 mEq/Kg established by Anvisa (Brasil, 2024). The saponification value (192.0 mg KOH/g) fell within the acceptable range (i.e., 188.0 to 196.0 mg KOH/g) according to AOCS (Firestone, 2006). The LO exhibited a short induction time of 2.2h according to the Rancimat method, confirming its susceptibility to oxidative processes. These findings indicate that the LO used in the present study meets quality standards and is suitable for use.

Characterization of NE

Macroscopic features

As shown in Figure S2 the NE-C exhibited a yellowish appearance, characteristic of LO; while the NE-0.25, NE-0.5, and NE-1.0 showed an increasingly brownish color as the GCE concentration increased. After the centrifugation test, none of the NEs showed signs of creaming, coalescence, or phase separation, thus confirming their intrinsic stability. in addition, all formulations displayed fluidity and translucence.

pH, Conductivity, and Rheology

From Table I it is possible to assert that all NEs had pH values within the range suitable for topical administration in the skin (i.e., 4.1-5.8) (Lambers et al., 2006; Proksch, 2018). When formulating skincare products, it is crucial for developers to carefully consider the skin pH to prevent potential skin irritation (Songkro et al., 2022). A slightly acidic pH is essential to inhibit the colonization of pathogenic bacteria (Lambers et al., 2006; Proksch, 2018). Moreover, since polyphenols are weakly acidic drugs, their penetration across the skin is enhanced as ionized moiety availability decreases (Hadgraft, Valenta, 2000; Akula, Lakshmi, 2018).

TABLE I
Physicochemical properties of cosmeceutical nanoemulsions containing linseed oil and Robusta green coffee extract

Nanoemulsion code	pH	Conductivity (μScm-1)	D50 (nm)	PDI	ZP (mV)
NE-C	5.2 ± 0.1	91.0 ± 1.4	79.6 ± 3.7	0.13	-26.1 ± 3.4
NE-0.25	5.0 ± 0.0*	389.5 ± 0.7*	79.7 ± 3.3	0.11	-29.2 ± 6.6
NE-0.5	5.2 ± 0.0	672.5 ± 3.5*	87.8 ± 5.6	0.20	-35.6 ± 5.4
NE-1.0	5.2 ± 0.2*	1235.0 ± 4.2*	82.2 ± 3.9	0.18	-38.5 ± 4.6

• NE-C, control nanoemulsion comprising only linseed oil (5% m/m); NE-0.25, nanoemulsion comprising linseed oil (5% m/m) combined with Robusta green coffee extract at 0.25% (m/m); NE-0.5, nanoemulsion comprising linseed oil (5% m/m) combined with Robusta green coffee extract at 0.5% (m/m); NE-1.0, nanoemulsion comprising linseed oil (5% m/m) combined with Robusta green coffee extract at 1.0% (m/m); D₅₀, mean droplet size; PDI, polydispersity index; ZP, zeta potential;
• ^*
  p < 0.05 compared to NE-C, one-way ANOVA followed by Tukey’s multiple comparison test.

Regarding conductivity, the high conductivity values obtained for the various NE ratify their o/w nature (Table I). The conductivity of NE-C was significantly lower than that of NE-0.25, NE-0.5, and NE-1.0 (p < 0.05). Of note, increasing the GCE loading in the NE from 0.0% to 1.0% significantly correlated with a linear rate increase (R² = 1.0) in conductivity. This indicates that the electrolytes containing the GCE are present in the continuous phase of the NE.

Figure 2 depicts the rheological properties of the developed NEs. Confirming the macroscopic analysis, NE-C, NE-0.25, NE-0.5, and NE-1.0 showed low viscosity, which remained unchanged with increasing shear and stress rates. The NE-C exhibited a k of 0.0014 Pa.s and n of 0.94 (R² = 0.98); for NE-0.25 the k was of 0.0004 Pa.s and the n was of 1.12 (R² = 0.99); for NE-0.5 the k was of 0.0014 Pa.s and the n was of 0.96 (R² = 0.99); and for NE-1.0 the k was of 0.0014 Pa.s and n was of 0.95 (R² = 0.99). Therefore, increasing the GCE content did not significantly affect the rheological properties of the NE. This Newtonian characteristic (i.e., n ≈ 1) makes them suitable for being administered as sprays, foams, or roll-ons (Clares et al., 2014). Factors such as droplet concentration, size distribution, and inter-droplet forces significantly influence the rheological behavior of NE (Polychniatou, Tzia, 2016).

FIGURE 2
Flow rheograms of cosmeceutical nanoemulsions comprising (A) only linseed oil (NE-C, control), or linseed oil combined with Robusta green coffee extract at (B) 0.25% m/m (NE-0.25), (C) 0.5% m/m (NE-0.5), and (D) 1.0% m/m (NE-1.0). A DIN spindle has been used. The samples were analyzed at 25 ± 2°C in three steps: 1) the shear rate increased from 0 to 700 s-1 (ascending curve), 2) the shear rate constant at 700 s-1, and 3) the shear rate decreased from 700 to 0 s-1 (descending curve). Each cycle lasted 3 minutes. Black symbols represent the upcurve and red symbols represent the downcurve.

Droplet Size, PDI, ZP, and Morphology

The results obtained by the DLS analysis are presented in Table I. NE-C, NE-0.25, NE-0.5, and NE-1.0 presented average D₅₀ values lower than 90 nm and PDI values ≤ 0.2. Adding GCE did not significantly affect the particle size or the polydispersity of the NE (p > 0.05).

Typically, the smaller the droplets the greater the Brownian motion, which enhances their ability to resist gravitational forces, thereby avoiding the tendency toward creaming or sedimentation (Bouchemal et al., 2004; Tadros et al., 2004). Additionally, their non-deformable characteristics hinder surface fluctuations, reducing the likelihood of droplet merging or coalescence (Tadros et al., 2004).

The PDI analysis provides insights into the dispersion quality of the sample. PDI values close to 1 indicate the presence of colloidal-sized globules with high polydispersity, suggesting uneven distribution of particle sizes. Conversely, PDI values close to zero indicate homogeneous particle size distribution (Yilmaz, Borchert, 2005; De Azevedo Ribeiro et al., 2015). It has been reported that PDI < 0.25 minimizes Ostwald ripening (Yilmaz, Borchert, 2005).

As shown in Figure S3, NE-C, NE-0.25, NE-0.5, and NE-1.0 all exhibited only one population of particles, confirming homogeneous droplet size distribution. A uniform distribution of particles also plays a crucial role in maintaining the stability of the NE. With heterogeneous particles in the system, the difference in Laplace pressure leads to the diffusion of molecules from small to large droplets through the continuous phase, resulting in Ostwald ripening (Yukuyama et al., 2016).

The ZP values were below -26.0, indicating the prevalence of negative charges in the several NEs and endorsing their kinetic stability. The GCE-loading also did not significantly affect the ZP (p > 0.05). The closer to zero the ZP, the greater the attractive forces between particles, leading to the destabilization of the colloidal dispersion system through aggregation (Roland et al., 2003). In contrast, the ZP range from |25 mV| to |30 mV| is suitable to establish an energy barrier and to prevent instability in pharmaceutic nanoparticles (Roland et al., 2003; De Azevedo Ribeiro et al., 2015). The negative charge of the NEs is likely attributed to: i) the presence of nonionic surfactants that form hydrogen bonds at the ether-oxygen of the polyoxyethylene chain, resulting in subsequent oxonium formation (Maruno, da Rocha-Filho, 2010); and ii) the occurrence of negatively ionized phytochemicals in either the GCE or the LO, such as phenolic acids, flavonoids, and PUFAs, which may also explain the slightly acidic pH of the NEs.

Results of the complementary TEM analysis presented in Figure 3 confirm the presence of regular and spherical structures in either NE-C or NE-0.25. The inner oily phase is homogenously dispersed without aggregates, suggesting that the surface charges of the particles are sufficient to maintain repulsion between them and consistent with the DLS findings.

FIGURE 3
Morphological features of cosmeceutical nanoemulsions comprising (A) only linseed oil (NE-C, control) or (B) linseed oil combined with Robusta green coffee extract at 0.25% m/m (NE-0.25). The grid was examined directly in the Transmission Electron Microscope at 80 kV and 25 ± 2°C. The scale is 0.2 μm and the magnification was 30000x.

Using a low-energy EPI method and the widely quoted surfactants Chremophor^® RH 40 and Span^® 80 (Jintapattanakit, Hasan, Junyaprasert, 2018) made it possible to obtain stable o/w NEs with suitable viscosity and evanescence. This method is based on the spontaneous transition in the curvature of the surfactant interfacial film resulting from composition changes in the system (Yukuyama et al., 2016). This condition is achieved by forming a bicontinuous or lamellar structure, which depends on the interfacial tension of the system, intrinsic physicochemical properties, and amount of surfactants to completely disperse the inner phase. As a result, kinetically stable emulsions with small particle sizes and homogeneous distribution are formed (Fernandez et al., 2004; Solans, Solé, 2012; Yukuyama et al., 2016).

The ultrasonic emulsification method developed LO-loaded NEs with atopic dermatitis treatment prospects. The formulated NEs exhibited a D₅₀ of 99.0 ± 1.1 nm, PDI value of 0.14 ± 0.02, and zeta potential of - 8.8± 0.03 mV (Kildaci et al., 2021). Another study (Sotomayor-Gerding et al., 2016) evaluated NEs prepared with LO using high-pressure homogenization and found that the average particle size in control emulsions ranged from 134 to 777 nm. A decrease in the particle size was observed with increasing homogenization pressure.

Although high-energy methods are widespread in lab-top cosmetic development, their industrial applicability is limited due to the high cost of manufacturing devices. Thus, low-energy methods like the one used in the current study are more practical and scalable (Bajerski et al., 2016; Azmi et al., 2019).

TPC and in vitro Antioxidant Activity of NE

The preparation of the GCE-loaded NEs resulted in a moderate reduction in TPC and antioxidant activity compared to the native extract, but a substantial proportion of the bioactivity was retained. The TPC values for NE-0.25, NE-0.5, and NE-1.0 were 94.1 ± 1.3 µg/g, 181.1 ± 6.3 µg/g, and 356.8 ± 13.5 µg/g, corresponding to 80.7 ± 7.3%, 77.7 ± 6.3%, and 76.6 ± 13.5% of the expected contents, respectively.

Consistent with these results, the reducing capacity values at 8.6 µg/mL for GCE, NE-0.25, NE-0.5, and NE-1.0 were 54.7 ± 0.5%, 48.2 ± 0.1%, 49.8 ± 0.3%, and 50.2 ± 0.1%, respectively. The DPPH radical scavenging activity of the NEs also showed a slight but statistically significant decrease compared to the native GCE (p < 0.05). Notably, a moderate positive correlation (R² = 0.75) was observed between the TPC and antioxidant activity, supporting the notion that the NE preparation process preserved most of the functional properties of the GCE.

These modest reductions could be attributed to the mechanical or thermal stress during NE preparation. However, based on existing evidence, thermal degradation is unlikely to be the main factor. A study reported that thermal degradation of phytochemicals in lyophilized GCE begins only at around 160°C (Grzelczyk et al., 2022), far exceeding the 75°C applied herein. Similarly, it has been demonstrated that LO exhibits excellent thermal stability, with residual fatty acid concentrations between 94-97% after 12 hours at 60°C, and lignans remain stable below 100°C, with significant degradation only at much higher temperatures (≈250 °C) (Spatari et al., 2019).

Moving forward, monitoring and optimizing critical process variables (e.g., such as stirring speed, duration, and temperature) will be essential to further enhance process reproducibility and product quality.

Preliminary Stability Test

The NEs underwent extreme storage conditions. All formulations maintained their organoleptic characteristics (aspect, color, and odor) and remained physically stable during the centrifugation test, not evidencing creaming or phase separation.

Table II shows that the pH values also remained unaltered. Exposure to light incidence and high temperatures can accelerate the oxidative degradation or triglyceride hydrolysis of emulsions containing vegetable oils, leading to a pH decrease due to the formation of fatty acids (Pereira et al., 2016). Therefore, the results indicate that the NE technology preserved the LO from undergoing degradation under the submitted storage conditions. This endorses the choice of this strategy for developing drug delivery systems based on edible oils.

TABLE II
The result of the preliminary stability test of cosmeceutical nanoemulsions containing linseed oil and Robusta green coffee extract underwent the heating-cooling cycle process

Nanoemulsion code	pH		Conductivity (μScm-1)		Antioxidant activity (%)
	Before	After	Before	After	Before	After
NE-C	5.2 ± 0.0	5.2 ± 0.0	83.9 ± 0.5	82.0 ± 0.1*	-	-
NE-0.25	5.0 ± 0.0	5.0 ± 0.0	403.4 ± 2.5	404.3 ± 2.1	39.4 ± 1.0	34.6 ± 0.8*
NE-0.5	5.1 ± 0.0	5.0 ± 0.0	683.0 ± 3.5	709.7 ± 0.6*	43.5 ± 0.7	36.7 ± 0.8*
NE-1.0	5.1 ± 0.0	5.1 ± 0.0	1234.3 ± 4.9	1310.0 ± 1.0*	45.7 ± 0.9	44.0 ± 1.3

• NE-C, control nanoemulsion comprising only linseed oil (5% m/m); NE-0.25, nanoemulsion comprising linseed oil (5% m/m) combined with Robusta green coffee extract at 0.25% (m/m); NE-0.5, nanoemulsion comprising linseed oil (5% m/m) combined with Robusta green coffee extract at 0.5% (m/m); NE-1.0, nanoemulsion comprising linseed oil (5% m/m) combined with Robusta green coffee extract at 1.0% (m/m);
• ^*
  p < 0.05 compared with respective nanoemulsion before the heating-cooling cycle process, Tukey’s T-test.

Following several heating-cooling cycles, the conductivity values of NE-C, NE-0.5, and NE-1.0 differed from those of their respective freshly prepared NEs (p < 0.05). However, no significant difference was found for NE-0.25. Assessing emulsion stability solely based on the electrical conductivity is not adequate, since the relationship between conductivity and instability is not always straightforward (Bernardi et al., 2011; De Azevedo Ribeiro et al., 2015).

The DPPH assay was used to assess the influence of heating-cooling cycles on the functional properties (antioxidant capacity) of NE-0.25, NE-0.5, and NE-1.0. As shown in Table II, the NE-1.0, unlike NE-0.25 and NE.0.5, did not show a significant difference in the antioxidant capacity after the sixth cycle. The loss of antioxidant activity was 12.2% and 15.6% for NE-0.25 and NE-0.5, respectively. The linear rate increase of the GCE loading in the NEs strongly correlated with a linear rate increase in the antioxidant activity at the end of the stability study (R² = 0.99). A moderate correlation (R² = 0.74) between the GCE loading and the remaining antioxidant activity of the NEs was also observed. Considering the correlations between TPC and antioxidant activity, it is reasonable that the degradation kinetics of the phenolic phytochemicals contained in GCE may depend on their concentrations. Further studies are required to confirm or refute this hypothesis.

Maintaining the stability of NE-based delivery systems during storage is crucial to ensure their quality, efficacy, and safety (Campanini et al., 2014). Irrespective of the GCE-loading, considering the high quantity of moieties possible for undergoing oxidation, these products should be sheltered from light and excessive heat while transporting and storing.

In vivo safety and efficacy

Safety assessment

An acute skin irritation test was conducted to confirm the safety of the developed NEs. The results presented in Figure 4 demonstrate that the components and concentrations used appear to be safe, since no signs of irritation, redness, swelling, inflammation, or any other abnormal changes in the skin were observed. These outcomes confirm good skin tolerability for topical application of all the developed NEs and their potential for use in skincare products.

FIGURE 4
Outcomes of acute irritation test following the treatment of hairless mice treated with several cosmeceutical nanoemulsions: (A) linseed oil (NE-C, control) immediately after application, (B) NE-C 72 h after application; (C) linseed oil combined with Robusta green coffee extract at 0.25% m/m (NE-0.25) immediately after application, (D) NE-0.25 72 h after application; (E) linseed oil combined with Robusta green coffee extract at 0.5% m/m (NE-0.5) immediately after application, (F) NE-0.5 72 h after application; (G) linseed oil combined with Robusta green coffee extract at 1.0% m/m (NE-1.0) immediately after application, and (H) NE-1.0 72 h after application.

Efficacy of NEs in protecting against UVB-induced damage to the skin

Figure 5 presents the results of the photoprotective efficacy of the developed NEs in decreasing inflammatory marker levels and restoring the skin’s antioxidant capacity following UVB exposure.

FIGURE 5
In vivo efficacy of cosmeceutical nanoemulsions comprising only linseed oil (NE-C, 0% GCE), or linseed oil combined with Robusta green coffee extract at 0.25% m/m (NE-0.25, 0.25% GCE), 0.5% m/m (NE-0.5, 0.5% GCE), and 1.0% m/m (NE-1.0, 1.0% GCE) against UVB-induced oxidate damage to the hairless mouse’s skin. (A) Evaluation of inhibition of neutrophil infiltration in skin exposed to UVB radiation. Effect on the recovery of skin’s antioxidant capacity exposed to UVB radiation by (B) FRAP and (C) ABTS assays. Inflammation and antioxidant potential were determined after 12 hours of irradiation. The bars represent the mean ± SEM of six mice per group and in two separate experiments. *p < 0.05 compared to the non-UVB-induced control (white bars); ^#p < 0.05 compared to the UVB-induced control (black bars), one-way ANOVA followed by Tukey’s test.

UVB irradiation significantly increased MPO activity compared to the non-UVB-induced group (Figure 5a). Treatment with NE-C and NE-0.25 significantly suppressed neutrophil migration by inhibiting MPO activity by 32% and 49%, respectively, compared to the UVB-induced control (Figure 5a). A statistically significant difference (p < 0.05) was observed between NE-C and NE-0.25. Neither the NE-0.5 nor the NE-1.0 significantly reduced MPO activity compared to the UVB-induced control and there was no statistically significant difference between these formulations. Since the combo of GCE (0.25% m/m) and LO (5.0% m/m) enhanced the latter in vivo anti-inflammatory activity in the skin by around 53%, rationally combining these nutraceuticals at this proportion has been shown to be feasible and effective.

The FRAP (Figure 5b) and ABTS (Figure 5c) assays demonstrated that UVB irradiation significantly decreased the skin’s antioxidant capacity compared to the non-UVB-induced group. In both assays, treatment with NE-0.25, unlike NE-C, NE-0.5, and NE-1.0 significantly inhibited the depletion of skin antioxidant activity compared to the UVB-induced control. Noteworthy, NE-0.25 reverted the UVB damage to the antioxidant balance in the skin to a level similar to the non-UVB-induced group. The absence of efficacy for NE-C suggests that the in vivo anti-inflammatory effect herein observed may not depend essentially on its antioxidant capacity.

Neither the MPO activity nor the skin’s antioxidant recovery capacity increased with increased GCE loading. Except for LO, the amounts of the phytochemicals increased 2-fold in NE-0.5 and 4-fold in NE-1.0 when administering the same amount of these formulations in comparison to NE-0.25.

Considering skin as a complex rate-controlled membrane (Bolzinger et al., 2012), saturation of its transport mechanisms might have been attained at the higher concentrations of the overall phytochemicals contained in the NE-0.5 and NE-1.0. Using natural skin permeation enhancers to overcome this limitation could be fully justified (Alhasso, Ghori, Conway, 2022). Moreover, at higher concentrations, the overall phytochemicals might have exerted an in vivo prooxidant effect on the skin (Zhou, Elias, 2013; Rajashekar, 2023). Despite being plausible, a more comprehensive analysis is required to confirm these hypotheses.

Robusta coffee has a broad range of industrial applications, from instant coffee production and caffeine extraction to use in cosmetics (mainly for treating cellulitis), biofuels, and biodegradable materials (Andrade, Perestrelo, Câmara, 2022). The versatility of LO, particularly its drying and emollient properties, makes it an important material in industries ranging from paints, coatings, linoleum flooring, printing inks, and plasticizers to conventional cosmetic products (Langyan et al., 2023). However, since using these industrial crops for innovating skincare cosmeceuticals is still incipient, the outcomes herein reported may be a starting point to support the manufacturer’s strategic planning switch toward the technological development of anti-aging products and sunscreens based on the combination of GCE and LO, thus aggregating even more value to their productive chain.

Study Limitations and Future Directions

The current study presents some limitations that warrant consideration when interpreting the findings. Although we performed a comprehensive quality control analysis confirming that the LO met regulatory standards with respect to the acid value, peroxide index, and saponification value, a full phytochemical profile (including quantification of fatty acids, lignans, sterols, tocopherols, and phenolics) was not conducted. Given that the phytochemical composition of LO can vary depending on agronomic (Dąbrowski et al., 2025) and processing factors (Spatari et al., 2019), such characterization would further strengthen the discussion of its potential biological contributions. Future studies should incorporate advanced analytical techniques to elucidate the specific phytochemical profile of the LO employed. This would enable a more precise correlation between composition and biological effects, thus enhancing the translational relevance of the findings.

While the physicochemical characterization of the NE demonstrated favorable attributes, such as an appropriate droplet size, polydispersity index, and stability under laboratory conditions, further studies are required to evaluate its long-term storage stability, photostability, and performance under varying environmental conditions. These additional assessments are essential to ensure the robustness and suitability of NEs for real-world applications (Rana et al., 2024).

The exclusive use of an in vivo acute irritation test to assess safety is also a limitation of this study. Future studies incorporating alternative methods, such as the Hen’s Egg Test on the Chorioallantoic Membrane (HET-CAM), would enhance the safety evaluation while aligning with the principles of the 3Rs (Replacement, Reduction, and Refinement) (Díaz et al., 2020).

Moreover, the absence of a treatment group receiving non-encapsulated GCE is noteworthy. This decision was intentional to prioritize the evaluation of the ability of the NEs to enhance topical delivery and photoprotective effects. The inclusion of a non-encapsulated GCE group would have increased the number of animals used, conflicting with ethical guidelines aimed at minimizing animal use. Furthermore, extensive literature demonstrates that phytochemicals, including GCE constituents, such as polyphenols and caffeine, exhibit limited skin permeability and, consequently, reduced bioavailability and efficacy when applied topically (Freire et al., 2019; Oliveira et al., 2022; Hammami et al., 2023; Reddy et al., 2024).

Nanocarrier-based delivery systems, such as the NEs evaluated here, have been shown to overcome these limitations by improving stability, penetration, and therapeutic outcomes (Freire et al., 2019; Romes, Abdul Wahab, Abdul Hamid, 2021). Therefore, our focus on encapsulated GCE aligns with current advances in the topical delivery of phytochemicals and supports the translational relevance of the findings. Nevertheless, we acknowledge that direct comparisons with non-emulsified GCE would provide complementary insights into the specific advantages conferred by NEs.

Although the NEs were tested in their liquid form in the current study to ensure uniform application and accurate dosing, future work should focus on incorporating the NEs into non-Newtonian semisolid delivery systems such as nanoemulgels to improve applicability, skin adherence, and patient compliance (Lal et al., 2023).

Performing in vitro release and in vitro skin penetration tests would be highly valuable for further characterizing the NEs. These studies could provide critical insights into the release kinetics of the bioactive compounds and their ability to penetrate the skin barrier, which are essential parameters for predicting in vivo performance. Additionally, such tests can guide optimization of the formulation to enhance drug delivery efficiency, stability, and therapeutic efficacy in topical applications (Santos, Swofford, Santiago, 2020; Shah et al., 2022; Lane, 2024).

Finally, although the low-energy emulsification method employed in this study was shown to be effective for producing stable NEs on a laboratory scale, future work should assess its scalability and reproducibility under industrial manufacturing conditions, to facilitate potential clinical and commercial translation.

CONCLUSION

The EPI method was prompt and affordable for obtaining stable cosmeceutical NEs comprising the nutraceuticals GCE and LO. The combination of GCE (0.25 % m/m) and LO (5.0 % m/m) provided in vivo photoprotective efficacy by decreasing inflammation markers and preserving the skin´s antioxidant activity following UVB-induced oxidative damage in an animal model. This synergic approach emerges as a safe and feasible strategy in developing skincare products, although there is still a need for additional studies to fully understand the role of this product in cutaneous photooxidative damage.

List of abbreviations:

ABTS, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid; ALA, α-linolenic acid; AOCS, American Oil Chemists’ Society; ANOVA, Analysis of Variance; Anvisa, Brazilian Health Regulatory Agency; CGAs, chlorogenic acids; 5-CQA, 5-caffeoylquinic acid; D₅₀, mean droplet size; DLS, Dynamic Light Scattering; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DTNB, 5,5’-Dithio-bis-(2-nitrobenzoic acid; EPI, Emulsion Phase Inversion; GCE, Robusta green coffee extract; HLB, Hydrophilic-Lipophilic Balance; HPLC, High-Performance Liquid Chromatography; HRP, horseradish peroxidase; HTAB, hexadecyltrimethylammonium bromide; LO, linseed (Linum usitatissimum L.) oil; MPO, myeloperoxidase; NE, nanoemulsion; o/w, oil-in-water; PDI, polydispersity index; PUFA, polyunsaturated fatty acids; ROS, reactive oxygen species; SD, standard deviation; SEM, standard error of the mean; TPTZ, 2,4,6-Tris(2-pyridyl)-s-triazine; TEM, Transmission Electron Microscopy; TFC, Total Flavonoid Content; TPC, Total Phenolic Content; UV, ultraviolet; ZP, zeta potential.

FUNDING

This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

INSTITUTIONAL REVIEW BOARD STATEMENT

All in vivo experimental procedures were performed according to the guidelines of the Animal Ethics Committee (O. Circ. CEEA No. 19/2020 on February 27th, 2020, process No. 110197.2020).

ACKNOWLEDGMENTS

We express our gratitude to Cia Iguaçu de Café Solúvel (Cornélio Procópio, PR, Brazil) for supplying and standardizing the dried green coffee extract. We also thank Mr. Pedro Ferreira de Oliveira from Federal University of São João del-Rei (UFSJ) for his assistance with the artwork.

SUPPLEMENTARY MATERIAL

Characterization of NE

Macroscopic features

The NE macroscopic features (appearance, creaming, and phase separation) were assessed in triplicate 24 hours after preparation. Afterward, the samples were centrifuged using a Baby^® I 206 BL centrifuge (Fanem^®, Guarulhos, SP, Brazil) at 3200 rpm for 30 minutes at 25 ± 2°C (Georgetti et al., 2006) and the macroscopic evaluation was repeated.

pH, Conductivity, and Rheology

The pH value of the NE was measured using a pH meter (Gehaka^®, São Paulo, SP, Brazil) calibrated with buffer solutions (pH 4.0 and 7.0). The NEs were diluted 1:10 (m/m) in purified water and measured at 25 ± 2°C (Georgetti et al., 2006).

The conductivity was determined using a HI 8819N conductivity meter (HANNA^® Instruments, Barueri, SP, Brazil), calibrated with a potassium chloride solution (0.1 N). The electrode was immersed directly in the NEs, and the conductivity was determined at 25 ± 2°C (Lonni et al., 2016).

The rheological properties of the NE were determined using an HR-2 Discovery Hybrid rotational rheometer (T.A. Instrument, New Castle, DE, USA) coupled with a DIN spindle. The samples were analyzed at 25 ± 2°C in three steps: 1) the shear rate increased from 0 to 700 s^-1 (ascending curve), 2) the shear rate constant at 700 s^-1, and 3) the shear rate decreased from 700 to 0 s^-1 (descending curve). Each cycle lasted for 3 minutes. The results presented the relationship between shear stress and shear rate.

The flow behavior index (n, dimensionless) and consistency coefficient (k, cP) values were determined by fitting the shear rate versus apparent viscosity data to the power law model using equation 1:

(1) $$\tau =k\cdot {\gamma }^n$$

where τ is the shear stress (Pa) and γ is the shear rate (s^-1). All tests were performed in triplicate.

DLS Analysis

The D₅₀ (µm) and PDI (dimensionless) were analyzed using a NanoPlus instrument (Nanosystems and Technologies GmbH, Meiningen, Germany). The ZP (mv) was determined by deriving the phoretic mobility measured with the same equipment. For D₅₀ and PDI analysis, the samples were diluted 1:100 (m/m) in purified water, while for zeta potential analysis, the samples were diluted 1:300 (m/m) in a potassium chloride solution (1 mM). All measurements were performed in triplicate at 25 ± 2°C with a 90° detection angle.

Morphology Assessment

TEM was employed to investigate the morphology of the NE. The formulations were diluted and applied onto a 400-mesh copper grid covered with a formvar/ carbon film. Uranyl acetate solution (2% m/v) was used for negative staining, and excess contrast was removed. The grid was examined directly in the TEM at 80 kV and 25 ± 2°C.

Evaluation of TPC and in vitro Antioxidant Activity of NE

The TPC in the NE was evaluated using the spectrophotometric method with the Folin-Ciocalteau reagent (Georgetti et al., 2006). Briefly, 0.3 g of NEs was added with 1.0 g of a 20% (m/v) KCl solution and 1500 μL of ethyl acetate. The samples were then vortexed for 3 min. Afterward, the tubes were centrifuged at 3600 rpm for 30 min, and the supernatant was carefully withdrawn from the remaining fraction. Both phases were then evaporated and resuspended in purified water, followed by the determination of TPC (Lonni et al., 2016).

The antioxidant activity of the NE was assessed using the DPPH free radical method (Georgetti et al., 2006; Casagrande et al., 2007). The NE was diluted in a 50% (v/v) hydroethanolic solution to achieve the IC₅₀ value previously determined for the GCE (i.e., 8.6 µg/ mL). Similarly, the GCE was also diluted in the same concentration as a reference. The tests were performed in triplicate. A solution of DPPH without the addition of NE was prepared as a positive control.

Preliminary Stability Test

The stability of the NE was evaluated through heating-cooling cycles following Anvisa guidelines (Brasil, 2004). The NEs were stored in semipermeable polypropylene containers at 4 ± 2°C for 24 h, followed by thawing at 40 ± 2°C for 24 h. This cycle was repeated six times, and the physicochemical properties (macroscopic features, pH, and conductivity) and the functional stability (antioxidant activity assessed by the DPPH method) were examined before initiating the test and after completing the final cycle.

FIGURES

FIGURE S1
Chromatographic profile of Robusta green coffee extract obtained by HPLC-UV. The Robusta green coffee extract was diluted in acetic acid (5%) aqueous solution (0.1 mg/mL), and the samples were filtered through a 0.22 µm pore size membrane. The injection volume was 25 µL. Chromatographic separations were carried out using a Spherisorb^® ODS-1 column (150 mm x 4.6 mm, 3 µm) from Waters Co. (Milford, MA, USA). The mobile phase comprised acetic acid (5%) aqueous solution (a) and acetonitrile (b) and the compounds were eluted at a 0.6 mL/min flow rate using the following gradient: 0-1 min (5% b) and 1-12 min (13% b). Detection was at (A) 320 nm for 5-CQA and (B) 272 nm for caffeine.

FIGURE S2
Macroscopic features of cosmeceutical nanoemulsions comprising (A) only linseed oil (NE-C, control), or linseed oil combined with Robusta green coffee extract at (B) 0.25% m/m (NE-0.25), (C) 0.5% m/m (NE-0.5), and (D) 1.0% m/m (NE-1.0) after 24 h of preparation.

FIGURE S3
The distribution droplet size of nanoemulsions comprising (A) only linseed oil (NE-C, control), or linseed oil combined with Robusta green coffee extract at (B) 0.25% m/m (NE-0.25), (C) 0.5% m/m (NE-0.5), and (D) 1.0% m/m (NE-1.0).

REFERENCES

Brasil. Ministério da Saúde. Agência Nacional de Vigilância Sanitária. Guia de Estabilidade de Produtos Cosméticos. 1st ed. Brasília, DF: 2004.

Casagrande R, Georgetti SR, Verri WA, Borin MF, Lopez RF V, Fonseca MJ V. In vitro evaluation of quercetin cutaneous absorption from topical formulations and its functional stability by antioxidant activity. Int J Pharm. 2007;328. https://doi.org/10.1016/j.ijpharm.2006.08.006.

Georgetti S, Casagrande R, Vicentini F, Verri W, Fonseca M. Evaluation of the antioxidant activity

of soybean extract by different in vitro methods and investigation of this activity after its incorporation in topical formulations. Eur J Pharm Biopharm. 2006;64. https://doi.org/10.1016/j.ejpb.2006.04.003.

Lonni AASG, Munhoz VM, Lopes GC, Longhini R, Borghi-Pangoni FB, dos Santos RS, et al. Development and characterization of multiple emulsions for controlled release of Trichilia catigua (Catuaba) extract. Pharm Dev Technol. 2016;21. https://doi.org/10.3109/10837450.2015.1081611.

DATA AVAILABILITY STATEMENT

Use of data not disclosed.

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DECLARATION OF INTEREST

The authors declare no conflict of interest. We certify that we have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements), or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

*

Correspondence: S. R. Georgetti, Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Estadual de Londrina, Londrina 86038-440, PR, Brasil; emails: [e-mail] [e-mail]

Associate Editor:

Guilherme Martins Gelfuso

Publication Dates

• Publication in this collection
  31 Aug 2026
• Date of issue
  2026

History

• Received
  30 Mar 2025
• Accepted
  10 July 2025