Marine Algae Extract-Loaded Nanoemulsions: A Spectrophotometric Approach to Broad-Spectrum Photoprotection

Abstract

The depletion of the ozone layer and climate change have increased exposure to ultraviolet (UV) radiation, driving the search for natural photoprotective agents. Marine macroalgae, particularly Gracilaria sp. (Rhodophyta) and Sargassum polyceratium (Ochrophyta), are rich in UV-absorbing bioactives, such as mycosporine-like amino acids (MAAs) and fucoxanthin, offering natural alternatives to synthetic sunscreens. This study aimed to develop and optimize a nanoemulsion incorporating both algal extracts, with MAAs and fucoxanthin strategically distributed in the aqueous and oil phases, respectively, to enhance synergistic broad-spectrum UV protection. MAAs were quantified in Gracilaria sp. using UHPLC-DAD, revealing 8.03 mg/g dry weight, primarily composed of shinorine and porphyra-334. Fucoxanthin was identified in S. polyceratium at 0.98 mg/g dry weight. A Box–Behnken design (BBD) was employed to optimize the nanoemulsion, targeting minimal droplet size and optimal ζ potential. The resulting formulation achieved a droplet size less than 100 nm and a ζ potential less than −25.0 mV. In vitro spectrophotometric analysis demonstrated significant photoprotective potential. The nanoemulsion containing only 375 ppm of algal extracts exhibited a UVA ratio of 1.25 and a critical wavelength of 379 nm, meeting the criteria for broad-spectrum protection and outperforming the commercial natural filter Helioguard^®365. These results confirm the efficacy of combining red and brown algae extracts in a nanoemulsion platform to deliver sustainable, low-dose photoprotection. This work presents, for the first time, the incorporation of red and brown algae extracts into a single nanoemulsion system, representing a novel strategy to maximize the combined photoprotective potential of MAAs and fucoxanthin. Ultimately, this investigation contributes to the growing field of marine-derived sunscreens and supports the advancement of “blue beauty” innovations aligned with eco-conscious formulation principles.

1. Introduction

Increasing levels of ultraviolet (UV) radiation—driven by factors such as climate change and stratospheric ozone depletion—have heightened the need for effective and sustainable photoprotective agents. In response, marine algae emerge as a promising source of molecules with unique properties to protect the skin from the damage caused by UV radiation. These organisms, which inhabit aquatic environments exposed to high levels of sunlight, have developed sophisticated biochemical mechanisms throughout their evolution to survive and thrive under conditions of high UV exposure. These mechanisms include the synthesis of photoprotective compounds, such as mycosporine-like amino acids (MAAs), carotenoids, sulfated polysaccharides, and polyphenolic compounds [1].

The genus Gracilaria, comprising red macroalgae (Rhodophyta), is widely distributed in marine and estuarine environments, spanning tropical to temperate regions [2]. Species within this genus are especially valued as a major source of agar—a sulfated polysaccharide extensively utilized in the pharmaceutical, biotechnological, and food industries. Among its bioactive compounds, shinorine and porphyra-334—two mycosporine-like amino acids (MAAs) isolated from Gracilaria cornea Agardh—have demonstrated remarkable photostability under UVB radiation and thermal stress, outperforming conventional pigments such as chlorophyll, carotenoids, and phycocyanin [3]. Additionally, other studies have shown that Gracilaria cornea can modulate the accumulation of UV-absorbing compounds, such as palythine, carotenoids, and phenolic compounds, in response to UVA and UVB radiation, suggesting photoreceptor-mediated photoprotection [4]. Furthermore, Gracilaria gracilis collected from Moroccan coasts has demonstrated significant photoprotective capacity through the presence of MAAs, such as shinorine, palythine–glutamine, and porphyra-334, with in vitro SPF values ranging from 5.55 to 9.34, depending on the extraction solvent, and notable anti-collagenase and anti-elastase activities [5]. In addition to UVB protection, MAAs also exhibit significant absorption in the UVA region [6]. Notably, these compounds are incorporated into commercial photoprotective products, such as Helioguard^®365—a natural sunscreen enriched with porphyra-334 and shinorine [7].

On the other hand, the genus Sargassum, a group of marine macroalgae belonging to the class Phaeophyceae (brown algae), is widely distributed along tropical and subtropical coastlines [8]. In recent years, the proliferation of Sargassum blooms has raised growing environmental concerns due to their negative ecological, economic, and social impacts. Consequently, alternative valorization strategies have been explored, including their use as cosmetic ingredients. Notably, organic extracts of S. muticum (Yendo) Fensholt have been shown to reduce reactive oxygen species (ROS) in UVB-irradiated cells, an effect attributed to their antioxidant properties and the upregulation of endogenous enzymes such as superoxide dismutase and catalase [9]. In addition, S. muticum, S. horneri, S. cristaefolium, and S. vulgare have demonstrated potent UV-absorbing and anti-aging properties, further reinforcing the photoprotective potential of the genus. For instance, S. muticum has been shown to accumulate UV-absorbing compounds, enhance non-photochemical quenching through the xanthophyll cycle, and boost antioxidant defenses to mitigate UV-induced photodamage, ultimately restoring photosynthetic performance after UV exposure [10]. Similarly, S. cristaefolium has demonstrated UVA-absorbing capacity and in vivo photoprotection, reducing epidermal thickening and preserving dermal collagen in UV-exposed mice [11]. Recent investigations on S. vulgare have demonstrated that this alga exhibited high photoprotective capacity across multiple UV-induced biological responses, achieving over 96% effective solar absorption radiation (ESAR) for vitamin D₃ synthesis, erythema, and immunosuppression at high concentrations and demonstrating strong potential as a cosmeceutical agent due to its antioxidant content and capacity to absorb harmful UV wavelengths [12]. Finally, S. horneri exhibits strong UV-protective activity by producing UV-absorbing compounds, increasing carotenoids, activating antioxidant enzymes, and enhancing photosynthetic recovery mechanisms, effectively mitigating UV-induced damage [13].

Moreover, studies employing electrical impedance spectroscopy (EIS) have demonstrated that formulations containing Sargassum cymosum help preserve the electrical resistance of skin membranes under UVB and oxidative stress conditions, reducing damage by up to 85% compared to untreated controls [14,15]. Additionally, Sargassum species contain bioactive polysaccharides with photoprotective potential—such as alginate and fucoidan—which have shown high in vitro SPF values of 32.218 and 65.709, respectively, depending on the concentration [16]. While these findings support the individual capabilities of red or brown algae in photoprotection, there remains a significant research gap regarding the development of integrated formulations that combine extracts from both Gracilaria and Sargassum to achieve synergistic, broad-spectrum UV protection.

Finally, nanoemulsions have emerged as advanced delivery systems in cosmetic and dermatological formulations due to their unique physicochemical properties and functional versatility. These colloidal dispersions, typically composed of oil, water, and surfactants, exhibit nanometric droplet sizes that confer superior stability, enhanced bioavailability of active ingredients, and improved skin penetration compared to conventional emulsions [17]. In the context of sun protection, nanoemulsions are particularly advantageous, as they enable the effective encapsulation and controlled release of photoprotective compounds, preserving their activity while minimizing degradation under UV exposure [14]. Their small droplet size also enhances the uniformity of topical application, leading to improved coverage and efficacy [18]. Moreover, by integrating natural antioxidant-rich extracts—such as those derived from marine algae—nanoemulsions can offer a dual function: physical protection against UVA and UVB radiation and biological defense via neutralization of reactive oxygen species (ROS) generated by solar exposure [17].

Therefore, considering the complementary photoprotective properties of Gracilaria sp. and Sargassum sp., particularly their content of mycosporine-like amino acids (MAAs) and fucoxanthin, this study aims to develop and optimize an oil-in-water nanoemulsion incorporating both extracts, each strategically allocated to distinct phases of the formulation based on the solubility of the active compounds: Gracilaria extract in the aqueous phase and Sargassum extract in the oil phase. This dual-phase design leverages the unique chemical characteristics and UV-absorbing capabilities of each extract, promoting a synergistic effect for enhanced broad-spectrum protection against both UVA and UVB radiation. By embedding these marine-derived bioactives into a nanometric delivery system, the proposed formulation seeks to provide a sustainable, eco-friendly alternative for next-generation sun care products in the cosmetic industry.

2. Materials and Methods

2.1. Algae Harvesting

Marine macroalgae were collected in Santa Marta (Magdalena, Colombia) in April 2022. The material was stored at −20 °C until use. To prepare the samples, the algae were washed with a saline solution, simulating seawater conditions, to remove fragments of other algae, marine animals, sand, and other debris. Subsequently, the material was evenly distributed into resealable plastic bags with small holes and stored again at −20 °C. Then, the algae were freeze-dried to obtain a dry weight (DW). These procedures were carried out protected from light.

The identification of the red macroalgae Gracilaria sp. (ID: JIW0005027) and Sargassum spp. was carried out by Dr. Briggite Gavio and Dr. Vanessa Urrea-Victoria based on morphological characteristics, following descriptions from the relevant literature. The algal nomenclature and taxonomy followed AlgaeBase [19]. Individual specimens of each sample were deposited at the JIWUKORI Algae Herbarium at Universidad Nacional de Colombia.

2.2. Extract Preparation

The extracts selected for this study were chosen based on previous results from the research group regarding photoprotective activity.

An aqueous extract of Gracilaria sp.: 3 g DW was weighed and placed in a flask with 60 mL of distilled water. Ultrasound-assisted extraction was performed for 15 min while keeping the bath refrigerated (in triplicate). The aqueous extract was then placed in a Falcon tube and frozen at −20 °C. Finally, the extract was freeze-dried to obtain the dry product [20].

A butanol fraction of Sargassum sp.: 3 g DW was weighed and placed in a flask with 60 mL of MeOH:water solution (1:1) (Merck, Darmstadt, Germany). Ultrasound-assisted extraction was performed for 15 min, keeping the bath refrigerated (in triplicate). The initial methanol:water extract was rotary-evaporated to remove the solvent. Then, to obtain the butanol fraction, the extract was dissolved in 30 mL of a BuOH:H₂O solution (1:1) (Merck, Darmstadt, Germany) and dried again by rotary evaporation [20].

The choice of extraction solvent was guided by the chemical nature of the target bioactive compounds in each algal species. For Gracilaria sp., an aqueous extraction was employed to isolate mycosporine-like amino acids (MAAs), such as shinorine and porphyra-334, due to their high water solubility and polar structure [21]. In contrast, a butanol fraction was used for Sargassum sp. to selectively extract fucoxanthin, a lipophilic carotenoid that exhibits greater solubility in medium-polarity organic solvents [22]. This solvent selection strategy ensured optimal recovery and preservation of the key photoprotective compounds, aligning with their physicochemical profiles and enhancing their functionality in the nanoemulsion system.

2.3. Extract Characterization

Aqueous extract of Gracilaria sp.: The identification and quantification of MAAs were developed according to the Urrea-Victoria et al. method, with modifications [21]. The analysis was performed using an Ultimate 3000 UHPLC system (Dionex, Thermo Scientific, Waltham, MA, USA). An aliquot of 5 μL of algae extract was injected onto a Synergi Hydro-RP 80A column (4 μm, 150 × 2.0 mm) (Phenomenex, Torrance, CA, USA) protected by a SecurityGuard pre-column. The flow rate was 0.3 mL.min⁻¹ at 22 °C. The gradient system combined an aqueous solution of 0.25% formic acid and 20 mM ammonium formate (solvent A) and acetonitrile (solvent B). It started with 100% A (0–3 min), followed by 5% A (3–7 min) and 50% A (7–8 min). The re-equilibration duration between individual runs was 7 min. Although a scan from 190 to 740 nm was performed, we focused on the set at 310, 320, 334, and 360 nm, as these wavelengths correspond to the peaks of the compounds of interest, such as MAAs. A porphyra-334 standard stock solution (>98% pure), prepared in-house and isolated by one of the authors (V. U-V.), was utilized in the calibration curve, and the results were expressed as mg MAAs.g 1 DW. 2.5. The corrected areas were then used to interpolate the concentrations based on the equation (y = 911918x + 4822.9; R² = 0.9993). The results were expressed as mg MAAs.g 1 DW. For shinorine (332 nm; 44,668 M¹ cm¹), the observed area was corrected using the respective molar extinction coefficient ratios, comparing them to those reported for porphyra-334 (334 nm; 42,300 M¹ cm¹).

Butanol fraction of Sargassum sp.: The fucoxanthin quantification in the butanol extract was determined using an analytical methodology employed by a research group. For this purpose, a Thermo Scientific Dionex Ultimate 3000 chromatograph equipped with a diode array detector (DAD) Dionex Ultimate 3000, a quaternary pump Dionex Ultimate 3000 RS, an online degasser, and an autosampler were used. A Phenomenex Kinetex C8 column (100 mm × 4.6 mm, 2.6 μm) was employed at 40 °C. The gradient system combined water (solvent A) and acetonitrile (solvent B). It started with 65% A (0–8 min), followed by 75% A (8–14 min). The re-equilibration duration between individual runs was 2 min. A 10 μL sample was injected, and detection was performed at a wavelength of 470 nm.

2.4. Sun Protection Factor (SPF), UVA Ratio (UVA-r), and Critical Wavelength (λc)

The absorbance values of the extracts (200 μL) were recorded by a UV–visible spectrophotometer (Varioskan LUX by Thermo Scientific, Waltham, MA, USA) with a spectral window from 290 to 700 nm (1 nm resolution/data point). Spectroscopic data were baseline-corrected.

The SPF of the obtained extracts and the developed product was evaluated using the Mansur method [23]. Stock solutions of the extracts were prepared at a concentration of 2 mg/mL in ethanol absolute pure 99.5% (EtOH; PanReac). Absorbance readings were taken between 290 and 400 nm, using EtOH as a blank. The SPF was calculated using the Mansur equation:

$$SPF=FC\times \sum EE\left(\lambda \right)320290\times I\left(\lambda \right)\times Abs\left(\lambda \right)$$

(1)

where FC corresponds to the correction factor value, which has a magnitude of 10; EE is a constant dependent on the wavelength and corresponds to the erythemal effect spectrum; I is the intensity of the solar spectrum at the wavelength of interest; and Abs is the measured absorbance of the solution at the specific wavelength.

Additionally, following the method described by Springsteen et al. [24], the UVA protection factor (UVA-r) and the critical wavelength (λc) were calculated. The UVA-r was determined as the ratio between the integrated intensities of the UVA (320–400 nm) and UVB (290–320 nm) regions. The λc, which indicates the wavelength at which 90% of the area under the absorbance curve (290–400 nm) is reached, was also calculated.

2.5. Nanoemulsion Preparation–Pseudoternary Diagram

Twenty-four nanoemulsions were prepared by varying the proportions of the dispersed phase (Capryol PGMC^®) (Gattefossé Co., Saint-Priest, France), the dispersing phase (deionized water), and the surfactant (Kolliphor EL^®:propylene glycol (3:2)) (Sigma Chemical Co., St. Louis, MO, USA) to evaluate the influence of composition on the formation of nanoemulsified systems and to identify the nanoemulsion region by a pseudoternary diagram. Sonication (at 80% power) (Q500-SONICATOR, Newton, MA, USA), was used as the preparation method, a high-energy technique suitable for generating emulsions with small droplet sizes. Capryol^® PGMC (Figure 1) is composed of propylene glycol monoesters and diesters of saturated caprylic and capric acids, offering amphiphilic properties that aid solubilization of lipophilic compounds. Kolliphor^® EL (also known as polyoxyl 35 castor oil) (Figure 1) consists of polyethoxylated castor oil, where 35 ethylene oxide units are added to castor oil, resulting in a nonionic surfactant with both lipophilic and hydrophilic domains. This structural configuration enhances emulsification and interfacial stability. Propylene glycol, a commonly used co-solvent, is included to modulate polarity and improve interfacial compatibility. These ingredients for the nanoemulsion were selected based on sustainable and eco-friendly criteria to develop a new cosmetic product.

All formulations were characterized in terms of droplet size and ζ potential after 1 day and 7 days of preparation using dynamic light scattering (DLS) with a Zetasizer Nano-ZS (scattering angle of 173°) (Malvern Instruments, Worcestershire, U.K). For ζ potential measurements, the nanoemulsions were prepared using 1% (v/v) deionized water. The analysis was performed on the same equipment using disposable folded capillary cells. All measurements were conducted at a temperature of 25 °C. Excel^® 16 was used to construct the pseudoternary diagram. MiniTab^®19 was used to generate the three-dimensional surface plots.

Day 1 measurements reflect the initial physicochemical characteristics immediately after preparation, including droplet size and ζ potential, which are critical parameters for defining the formation and uniformity of the nanoemulsion. Day 7 measurements were chosen to assess the formulation’s short-term physical stability, particularly to observe potential changes due to surfactant redistribution, interfacial reorganization, or droplet coalescence. This timeframe aligns with previously reported studies on nanoemulsion stabilization dynamics and allows for the detection of early trend behavior [25,26].

2.6. Nanoemulsion Optimization

Once the surfactant and oil levels were selected based on the results of the pseudoternary phase diagram, the optimal emulsification conditions for incorporating the algae extract were determined using a Box–Behnken design (BBD) [14,15]. The response variable for optimization was droplet size.

2.7. Statistical Analysis

The experimental design, data analysis, and nanoemulsion optimization were performed with Minitab^® software (version 19, State College, PA, USA).

3. Results and Discussion

3.1. Extract Characterization

Chromatographic analysis by HPLC-DAD confirmed the presence of mycosporine-like amino acids (MAAs) in Gracilaria sp. and fucoxanthin in Sargassum sp. (Figure 2). In Gracilaria sp., the total MAA content was quantified at 8.03 ± 0.009 mg/g dry weight (DW), with shinorine and porphyra-334 contributing 4.23 ± 0.007 mg/g DW and 3.80 ± 0.002 mg/g DW, respectively. These findings are consistent with previously reported MAA levels in red algae [27,28,29,30,31], supporting their recognized potential as natural photoprotective and antioxidant agents.

In Sargassum sp., the butanol extract exhibited a fucoxanthin concentration of 0.98 ± 0.005 mg/g DW—substantially higher than values typically reported for other solvent extracts of this species (0.3–0.7 mg/g). This indicates that the butanol fraction may be particularly effective in concentrating this UV-protective carotenoid [32,33,34].

MAAs, such as shinorine and porphyra-334, are hydrophilic compounds known for their strong UVA absorption, photostability, and antioxidant effects, making them promising candidates for natural sunscreen formulations. These results corroborate earlier studies who demonstrated that topical application of MAAs from red algae could reduce photoaging and perform comparably to synthetic UVA filters [35,36]. Similarly, fucoxanthin in Sargassum sp. contributes to UVB protection and oxidative stress reduction, further validating its utility in marine-derived cosmetic formulations [37,38,39].

3.2. Nanoemulsion Preparation–Pseudoternary Diagram

Nanoemulsions (

Table 1. Droplet size and ζ potential for emulsions without Gracilaria sp. and S. polyceratium extracts.

No	Oil (%)	Smix (%)	Water (%)	Day 1		Day 7
				Droplet Size (nm)	Potential ζ (mV)	Droplet Size (nm)	Potential ζ (mV)
1	4	15	81	17.8 ± 0.7219	−24.13 ± 0.83	17.46 ± 0.08	−23.92 ± 0.24
2	4	20	76	27.36 ± 4.05	−24.14 ± 0.20	32.38 ± 5.16	−23.88 ± 0.36
3	4	25	71	54.27 ± 2.77	−22.82 ± 0.32	21.70 ± 1.36	−22.78 ± 0.31
4	4	30	66	18.62 ± 0.3814	−23.56 ± 0.18	15.71 ± 0.14	−23.84 ± 0.16
5	4	35	61	16.88 ± 0.1943	−23.24 ± 0.28	15.09 ± 0.09	−22.84 ± 0.31
6	8	10	82	17.59 ± 0.4099	−22.86 ± 0.13	26.72 ± 0.21	−22.42 ± 0.19
7	8	15	77	172.5 ± 1.249	−23.31 ± 0.10	253.40 ± 5.99	−23.56 ± 0,07
8	8	20	72	18.15 ± 0.07937	−23.99 ± 0.18	14.67 ± 0.03	−23.68 ± 0.42
9	8	25	67	333.9 ± 13.1	−23.32 ± 0.16	280.40 ± 2.54	−22.88 ± 0.17
10	8	30	62	31.82 ± 0.3584	−24.07 ± 0.17	21.29 ± 0.03	−24.24 ± 0.12
11	8	35	57	78.29 ± 7.053	−24.12 ± 0.13	22.14 ± 0.03	−24.68 ± 0.16
12	12	10	78	20.75 ± 0.9283	−23.38 ± 0.21	18.31 ± 0.15	−22.70 ± 0.32
13	12	15	73	322.5 ± 3.584	−24.13 ± 0.05	289.20 ± 3.70	−22.80 ± 0.32
14	12	20	68	478.1 ± 5.424	−24.08 ± 0.10	340.50 ± 5.78	−24.46 ± 0.06
15	12	25	63	316.1 ± 2.572	−23.75 ± 0.22	393.70 ± 1.58	−24.58 ± 0.16
16	12	30	58	359.8 ± 3.786	−23.08 ± 0.34	124.90 ± 10.54	−24.62 ± 0.11
17	12	35	53	949.9 ± 3.01	−24.08 ± 0.06	210.30 ± 17.68	−24.16 ± 0.13
18	16	10	74	394.6 ± 2.542	−23.4 ± 0.02	378.20 ± 11.82	−22.44 ± 0.75
19	16	15	69	380.6 ± 3.118	23.05 ± 0.26	330.30 ± 2.76	−25.60 ± 0.29
20	16	20	64	439.3 ± 3.464	−23.23 ± 0.38	399.60 ± 0.93	−20.23 ± 0.18
21	16	25	59	390.8 ± 3.404	−23.59 ± 0.38	342.50 ± 3.46	−22.12 ± 0.09
22	20	10	70	472.1 ± 5.897	−20.09 ± 0.08	420.10 ± 1.78	−24.44 ± 0.03
23	20	15	65	258.4 ± 11.17	−25.54 ± 0.35	262.30 ± 10.50	−23.96 ± 0.17
24	20	20	60	590.2 ± 4.576	−24.2 ± 0.33	435.30 ± 6.47	−23.86 ± 0.12

Oil: Capryol PGMC^®; S_mix: Kolliphor EL^®: propylene glycol (3:2).

) were prepared by varying the proportions of the dispersed phase (Capryol^® PGMC), the dispersing phase (deionized water), and the surfactant–cosurfactant mixture (S_mix). Emulsification was carried out using an ultrasonicator (Q500-SONICATOR, Newton, MA, USA) operating at 80% amplitude for 4 min.

The droplet sizes of the prepared nanoemulsions ranged from 16.88 ± 0.19 nm (formulated with 4% oil, 35% S_mix, and 61% water) to 590.2 ± 4.58 nm. For the construction of the pseudoternary phase diagram, only formulations with droplet sizes below 100 nm—measured at both 1 day and 7 days post-preparation—were considered. This selection ensured that only systems meeting the criteria for true nanoemulsions and demonstrating resistance to coalescence were included. The nanoemulsion region was identified within the compositional space defined by 4–12% oil, 10–35% S_mix, and 57–82% water. Based on these data, the pseudoternary diagram was constructed to illustrate the nanoemulsion-forming region (Figure 3).

On the other side, an unexpected yet noteworthy observation in this study was the significant reduction in droplet size of some emulsions after seven days of standing. This phenomenon, although uncommon, may be explained by the progressive reorganization and stabilization of the interfacial film, leading to tighter droplet packing and improved surfactant alignment [24,25]. Such delayed stabilization is characteristic of systems with high HLB surfactants and co-solvents, which continue to redistribute over time, reducing interfacial tension and promoting smaller, more stable droplets [40,41]. This maturation effect enhances the homogeneity of the nanoemulsion and may improve long-term stability, aligning with findings in similar lipid-based formulations [42].

3.3. Nanoemulsion Optimization

Based on the results obtained in the pseudoternary diagram, the levels for the experimental statistical design were selected (

Table 2. Matrix of independent variables of the BBD.

Independent Variables	Levels
	−1	0	−1
Oil (%)	4	8	12
Smix (%)	15	30	45
Sonication time (min)	2	5	8

Oil: Capryol PGMC^®; S_mix: Kolliphor EL^®: propylene glycol (3:2).

).

To evaluate the variation in droplet size and potential ζ of the nanoemulsions as a function of the formulation variables (%oil, %S_mix, and sonication time), the surface models were used. As shown in

Table 3. Factor points, independent factor levels, and experimental values of dependent variables for the Box–Behnken design (BBD).

No	Oil (%)	Smix (%)	Water (%)	Sonication Time (min)	Day 1		Day 7
					Droplet Size (nm)	Potential ζ (mV)	Droplet Size (nm)	Potential ζ (mV)
1	8	30	62	5	45.72 ± 3.39	−17.2 ± 2.0	52.56 ± 6.92	−23.0 ± 2.8
2	8	30	62	5	34.70 ± 8.80	−24.0 ± 0.7	41.50 ± 2.30	−24.5 ± 0.6
3	12	30	58	8	30.09 ± 0.47	−22.6 ± 3.0	33.92 ± 1.15	−19.9 ± 2.1
4	4	45	51	5	38.77 ± 0.78	−26.1 ± 0.9	38.32 ± 3.96	−25.0 ± 3.1
5	4	15	81	5	29.33 ± 2.55	−23.8 ± 3.2	89.80 ± 11.88	−13.4 ± 0.6
6	8	30	62	5	40.12 ± 2.01	−24.8 ± 3.2	23.44 ± 0.51	−18.4 ± 3.0
7	8	30	62	5	55.96 ± 7.25	−21.6 ± 1.7	26.60 ± 0.21	−24.1 ± 2.7
8	8	45	47	8	31.26 ± 2.71	−9.3 ± 0.8	46.17 ± 0.82	−11.3 ± 1.7
9	8	15	77	8	78.01 ± 0.38	−20.4 ± 2.0	125.70 ± 8.58	−24.8 ± 0.9
10	8	45	47	2	40.73 ± 3.49	−18.0 ± 0.5	30.95 ± 1.32	−15.4 ± 3.5
11	12	30	58	2	27.04 ± 0.51	−21.4 ± 1.2	24.25 ± 0.73	−16.5 ± 2.4
12	8	30	62	5	50.96 ± 2.24	−15.2 ± 2.0	31.56 ± 0.14	−22.5 ± 0.4
13	4	30	66	2	43.42 ± 9.93	−33.4 ± 2.1	22.21 ± 0.51	−18.1 ± 2.7
14	12	45	43	5	55.26 ± 0.87	−11.8 ± 1.4	33.09 ± 0.62	−11.7 ± 1.6
15	8	30	62	5	41.19 ± 1.36	−18.9 ± 1.4	29.99 ± 0.73	−18.5 ± 2.3
16	8	15	77	2	28.98 ± 0.72	−29.9 ± 3.5	25.41 ± 0.33	−20.9 ± 3.2
17	12	15	73	5	55.78 ± 0.43	−22.3 ± 2.4	89.94 ± 4.32	−19.0 ± 2.3
18	4	30	66	8	26.34 ± 1.93	−31.4 ± 0.9	32.14 ± 2.25	−14.9 ± 1.3

Oil: Capryol PGMC^®; S_mix: Kolliphor EL^®: propylene glycol (3:2).

, eighteen points of the BBD matrix represent the different combinations of the independent variables and the responses of droplet size and potential ζ.

The experimental data were used to calculate the quadratic polynomial models. The equations are presented below:

Droplet size (nm) = 10.0 + 10.61 Time + 0.87 Oil(%) − 0.51 S_mix(%) − 0.407 Time * Time − 0.226 Oil(%) * Oil(%) + 0.0235 S_mix(%) * S_mix(%) + 0.761 Time * Oil(%) − 0.3250 Time * S_mix(%) + 0.0418 Oil(%) * S_mix(%)

(2)

Potential ζ (nm) = −48.6 + 5.72 Time + 4.57 Oil(%) − 0.989 S_mix(%) − 0.351 Time * Time − 0.235 Oil(%) * Oil(%) + 0.02463 S_mix(%) * S_mix(%) − 0.067 Time * Oil(%) − 0.0378 Time * S_mix(%) + 0.0117 Oil(%) * S_mix(%)

(3)

Accordingly, analysis of variance (ANOVA) was used to determine the statistical significance of the independent variables and their interactions on the response variables—droplet size and ζ potential. The suitability of the models was evaluated using the coefficient of determination (R²) and the p-value for the lack-of-fit test (p-value > 0.05). The statistical analysis of the nanoemulsion optimization is summarized in

Table 4. Analysis of variance of a surface response of quadratic equations for droplet size and potential ζ.

Variable		Droplet Size		Potential ζ
		F-Value	p-Value	F-Value	p-Value
Linear terms	A	8.61	0.019	1.28	0.291
	B	9.84	0.014	5.35	0.049
	C	3.81	0.087	16.86	0.003
Quadratic terms	A2	0.84	0.386	2.63	0.143
	B2	0.82	0.391	3.73	0.089
	C2	1.76	0.221	8.11	0.022
Interaction terms	AB	4.80	0.060	0.15	0.704
	AC	12.30	0.008	0.70	0.427
	BC	0.36	0.564	0.12	0.739
Lack-of-fit		0.37	0.778	1.34	0.360

A: time of sonication (min); B: Oil(%); C: S_mix(%). In the model, values of p <0.05 are considered significant.

. The results indicated that the quadratic model adequately explained the behavior of the system concerning both droplet size and ζ potential. For droplet size, the model demonstrated significant predictive power, with a p-value < 0.05 for the oil concentration and S_mix, while sonication time approached significance. Among the interaction terms, the interaction between oil and sonication time (AC) was significant, suggesting that the combined influence of these variables strongly impacts droplet size.

For ζ potential, S_mix and sonication time were significant linear contributors (p = 0.049 and p = 0.003, respectively). Additionally, the quadratic term for S_mix (C²) showed significance (p = 0.022), highlighting its nonlinear effect on system stability. Again, the lack-of-fit was not significant (p = 0.360), supporting the model’s adequacy.

Overall, these results suggest that oil and S_mix concentrations as well as their interactions with sonication time play pivotal roles in defining both the droplet size and the electrostatic stability of the nanoemulsion. The model demonstrated good explanatory power, providing a robust basis for formulation optimization.

Three-dimensional surface plots were generated by MiniTab^®19 for the response variables (droplet size and ζ potential) of the nanoemulsions, evaluating the effect of two independent variables and one other variable that remained constant at the center point (Figure 4 and Figure 5).

Finally, the three factors were optimized to obtain the nanoemulsion with the smallest droplet size and ζ potential. Based on the optimization of the nanoemulsion response variables, the emulsion composition is oil 4%, S_mix 20.8%, and sonication time 2 min (

Table 5. Final characteristics of the optimized formulation.

Response Variable	Experimental Value
Droplet size (nm)	32.42
Potential ζ	−26.0
Final composition
Kolliphor EL®: propylene glycol (%)	20.8%
Capryol PGMC (%)	4%
Deionized water (%)	73.7%

Oil: Capryol PGMC^®; S_mix: Kolliphor EL^®: propylene glycol (3:2).

).

3.4. Sun Protection Factor (SPF), UVA Ratio (UVA-r), and Critical Wavelength (λc)

The photoprotective potential of the formulations was evaluated through SPF, UVA protection factor (UVA-r), and critical wavelength (λc) measurements (Figure 6). As expected, benzophenone-3 exhibited the highest SPF value, reaffirming its well-established efficacy as a UVB filter. Among the algal extracts, Gracilaria sp. demonstrated superior photoprotection compared to Sargassum sp., as reflected in both SPF and UVA-r values. Notably, nanoemulsions containing 150 ppm and 375 ppm of the combined algal extracts outperformed the commercial natural filter Helioguard^®365 (100 ppm) (Mibelle Biochemistry, Buchs, Switzerland), indicating a potential synergistic effect within the formulation.

The aqueous extract of Gracilaria sp. exhibited the highest UVA-r value (2.13) and a critical wavelength (388 nm), suggesting strong UVA absorption and photostability. In comparison, the butanolic extract of Sargassum sp. showed a UVA-r of 1.80 and a λc of 385 nm—both within the threshold for broad-spectrum protection (λc ≥ 370 nm). Among the formulations, the nanoemulsion with 375 ppm of bioactives achieved a UVA-r of 1.25 and a λc of 379 nm, confirming its moderate but effective UVA coverage. By contrast, the positive control benzophenone-3 yielded a UVA-r of 0.98 and λc of 350 nm, underscoring its UVB-focused photoprotective action [43].

The combined use of both algal extracts in a single nanoemulsion provided complementary and synergistic photoprotection. MAAs from Gracilaria sp. primarily contributed to UVA absorption and ROS neutralization [7,44], while fucoxanthin from Sargassum sp. extended protection to the UVB range and enhanced antioxidant responses [45,46]. This dual mechanism supports the phase-specific formulation strategy: incorporating Gracilaria extract in the aqueous phase and Sargassum extract in the oil phase.

In vitro spectrophotometric analysis confirmed that, even at a low total concentration (375 ppm), the nanoemulsion delivered broad-spectrum protection (UVA-r > 0.7; λc = 379 nm). These results align with previous studies, such as Galani et al. (2023), which demonstrated that antioxidant-loaded nanoemulsions can enhance the SPF of topical products even at low concentrations [17].

Beyond UV filtration, this formulation is expected to provide biological skin protection. A recent study using Sargassum cymosum-based nanoemulsions showed up to 85% preservation of skin membrane integrity under UVB exposure, as measured by electrical impedance spectroscopy (EIS) [14]. These findings reinforce the potential of algae-derived compounds to mitigate oxidative and structural damage caused by solar radiation.

While the SPF values achieved may be modest compared to synthetic filters, the effectiveness at low concentrations combined with the natural origin, biodegradability, and reef-safe profile of the ingredients represent a valuable trade-off. This approach aligns with current trends in environmentally conscious cosmetics [47]. Moreover, the nanoemulsion platform offers scalability and formulation flexibility, supporting further SPF enhancement through extract concentration adjustment or the incorporation of other natural boosters [48,49].

These results validate the use of Gracilaria sp. and Sargassum sp. extracts in advanced, eco-friendly photoprotective formulations. This study contributes to the growing field of marine-derived sun care technologies by integrating biodiversity, nanotechnology, and sustainable formulation science.

Future work should aim to expand both the biological and physicochemical evaluation of the developed nanoemulsion. In vitro assays using UV-exposed skin cell models are needed to confirm the formulation’s protective effects at the cellular level, including potential anti-inflammatory and antioxidant responses. Additionally, further physicochemical characterization—such as rheological behavior, texture profile analysis, long-term stability studies, and skin permeation assays—will provide a more comprehensive understanding of the formulation’s performance and applicability. These complementary studies will be essential to support the positioning of algae-based nanoemulsions as effective, stable, and sustainable photoprotective alternatives for cosmetic applications.

4. Conclusions

This study demonstrated the development of a nanoemulsion incorporating Gracilaria sp. and Sargassum sp. extracts as a sustainable approach for broad-spectrum UV protection. MAAs and fucoxanthin, identified in the respective algae, were effectively combined in an optimized oil-in-water nanoemulsion with a droplet size of 32.42 nm and ζ potential of −26.0 mV. Photoprotective evaluation showed that the formulation, even at a low concentration (375 ppm), provided broad-spectrum coverage (UVA-r = 1.25; λc = 379 nm), surpassing the performance of a commercial natural filter. These findings highlight the synergistic potential of red and brown algae in nanoformulations for eco-friendly sun care products. This work contributes to the expanding field of marine-derived photoprotectants, offering a promising alternative aligned with “blue beauty” and environmentally conscious formulation strategies. Future studies should focus on increasing extract concentrations and validating in vivo efficacy for cosmetic applications.