Exploring the Potential of Extracts from Sloanea medusula and S. calva: Formulating Two Skincare Gels with Antioxidant, Sun Protective Factor, and Anti-Candida albicans Activities

Sloanea is a plant genus, native to tropical regions, used in medicinal practices for its anti-inflammatory properties. This study aimed to determine the antioxidant activity, sun protective factor (SPF), and antifungal of extracts obtained from two species of Sloanea and to develop extract-based gels with antioxidants, photoprotective, and anti-Candida albicans effects. Ethanolic extracts from S. medusula and S. calva collected in Chocó, Colombia, were used for antioxidant activity and SPF determination using the DPPH assay and the Mansur equation, respectively. Extracts were characterized using HPLC-MS and used to prepare the gels. The viscosity of the extract-based gels was evaluated using an MCR92 rheometer. In addition, the anti-Candida activity of extracts against five yeasts and anti-C. albicans of gels were evaluated following the Clinical and Laboratory Standards Institute M27, 4th Edition. High DPPH radical scavenging activity (42.4% and 44.7%) and a high SPF value (32.5 and 35.4) were obtained for the extracts of S. medusula and S. calva, respectively. Similarly, extract-based gels showed significant DPPH radical scavenging activity of 54.5% and 53.0% and maximum SPF values of 60 and 57. Extract from S. medusula showed an important antifungal activity against C. albicans (minimal inhibitory concentration (MIC) of 2 µg/mL). In contrast, S. calva extract was active against C. krusei, C. albicans (MIC of 2 µg/mL) and C. tropicalis (MIC of 4 µg/mL). Sloanea medusula gel (0.15%) exhibited an important C. albicans growth inhibition (98%), while with S. calva gel (0.3%) growth inhibition was slightly lower (76%). Polyphenolic and triterpenoid compounds were tentatively identified for S. medusula and S. calva, respectively. Both extracts can be considered promising sources for developing photoprotective gels to treat skin infections caused by C. albicans.


Introduction
Throughout history, plant extracts have played a significant role in medicinal and cosmetic formulations [1,2]. Unfortunately, these valuable natural ingredients have gradually been substituted with chemicals, which have had a negative global impact, especially on ecosystems [3] and skin microflora [4]. Today, diverse strategies are being implemented to mitigate this impact and promote enhanced environmental consciousness. One of these strategies involves promoting natural extracts rich in antioxidant compounds as active ingredients as these represent a sustainable and eco-friendly alternative for pharmaceutical  Figure S1 shows the extracted positive and negative ion chromatograms of the compounds present in the crude extracts. Tables 1, 2, and Supplementary Table S1 summarize the relevant data from the HPLC-MS analysis of the ethanolic extract of S. medusula and S. calva, respectively. The identified tentative molecules, possible fragment ions, neutral losses, and mass spectra are shown in Supplementary Figures S2-S8. The DPPH radical scavenging activity of the ethanolic extracts obtained from S. medusula and S. calva was evaluated using the DPPH assay at a concentration of 0.7 mg/mL. The results were expressed as the percentage of DPPH radical scavenging activity and Trolox equivalent antioxidant capacity (TEAC) ( Table 3). As shown in Table 3, the extract of S. medusula showed significant radical scavenging activity (42.4%); however, the extract of S. calva showed slightly higher activity (44.7%). Moreover, the anti-radical efficacy expressed as TEAC showed values of 1.1 and 2.7 mM Eq. Trolox/100 g sample weight for the extracts of S. medusula and S. calva, respectively.  . Results were expressed as the mean ± standard error of the mean from three independent assays.

Determination of in vitro Sun Protective Factor of Sloanea Extracts
The determination of SPF in vitro for the two Sloanea species evaluated is shown in Table 4. SPF values were evaluated at three concentrations, which ranged from 0.25 to 0.75 mg/mL, following the increasing order indicated below, S. medusula < S. calva. According to these results, at the concentration of 0.75 mg/mL, both evaluated extracts provide high protection against UVB radiation: S. medusula (SPF 32.5) and S. calva (SPF 35.4). Results were expressed as the mean ± standard error of the mean from three independent assays.
Additionally, as a complement to SPF, three photoprotective parameters were evaluated following the methodology employed by Caballero-Gallardo et al. [29]. Values of the UVA/UVB ratio, the transmission of erythema (%), and the transmission of pigmentation (%) showed the photoprotective potential of both extracts. Results are shown in Supplementary Table S2.

Evaluation of the Anti-Candida Activity of Sloanea Extracts
Sloanea medusula and S. calva were evaluated at 10 concentrations ranging from 0.5 to 256 µg/mL to determine their MICs against C. krusei ATCC 6258 (L6), C. auris Ca17 (L25), C. tropicalis ATCC 200956, C. glabrata LMDM 34 (L7), and C. albicans ATTC 10231. The results, expressed as the geometric mean of the minimum inhibitory concentration (GM-MIC), are shown in Table 5. It can be observed that these yeasts have different sensibility profiles against two extracts evaluated, anti-C. albicans being potentially the most remarkable with an MIC of 2.0 µg/mL for both extracts, followed by S. calva against C. krusei (MIC of 2.0 µg/mL), S. calva against C. tropicalis (MIC of 4.0 µg/mL), and S. medusula against C. auris (MIC of 16.0 µg/mL). In addition, C. krusei ATCC 6258 (L6) was used as a control strain, and its susceptibility was evaluated using itraconazole (ITC) and amphotericin B (AMB), which showed MIC values of 0.125 µg/mL and 0.630 µg/mL, respectively. Both the S. medusula (0.3%) and the S. calva (0.3%) gels showed a homogeneous appearance, without signs of lumps or precipitation. The S. medusula gel exhibited a terracotta color ( Figure 1A) and a characteristic aroma of pleasant intensity, reminiscent of burnt grass, while the S. calva gel showed an ochre color ( Figure 1B) and a slightly mentholated odor. The coloration of each gel was provided by the same plant extracts.

Rheology and Extensibility of Sloanea-Extract-Based Gels
Viscosity is frequently employed to analyze the characteristics of a formulation and ensure optimal distribution. It also enables formulators to enhance the efficacy of personal care products because this can be adjusted according to specific application needs [30].

Rheology and Extensibility of Sloanea-Extract-Based Gels
Viscosity is frequently employed to analyze the characteristics of a formulation and ensure optimal distribution. It also enables formulators to enhance the efficacy of persona care products because this can be adjusted according to specific application needs [30].
In this study, the viscosity (Pa·s) of the gels was analyzed as a function of the shear rate (1/s) to monitor the stability of the formulation and define its rheological behavior Figure 2 shows that the limiting low-shear viscosity of S. calva gel is higher in comparison with S. medusula gel. This is due to a higher concentration of the rheologic modifier (Car bopol ® Ultrez 21) in the S. calva formulation (Section 4.7). It is observed that the shear viscosity of the formulations decreases with increasing shear rate after a sufficiently large rate is attained. The descending part of the flow curve confirmed that both formulations exhibited non-Newtonian viscosity, i.e., pseudoplastic behavior [31]. The measurement of the extensibility area provides information about the defor mation threshold of the formulations [32]. The extensibility area was measured by observ ing the increase in the surface of the gels under progressively increasing pressures (0.5958 g, 1.1624 g, and 4.2465 g) at 1 min intervals. This analysis resulted in the determination o extensibility area values, which provide valuable information about the capacity of S. me dusula-and S. calva-based gels to stretch over the skin surface without experiencing break age. Under these conditions, the S. medusula-based gel exhibited an extensibility area o 10.78 ± 2.5 cm 2 , showing a comparable elasticity or flexibility to the S. calva-based gel with an extensibility area of 10.56 ± 3.1 cm 2 .

Efficacy Studies
Evaluation of the Antioxidant Activity of Extract-Based Gels by DPPH Assay The results of antioxidant activity expressed as DPPH radical scavenging activity (% and TEAC of gels based on S. medusula and S. calva extracts, respectively, are shown in In this study, the viscosity (Pa·s) of the gels was analyzed as a function of the shear rate (1/s) to monitor the stability of the formulation and define its rheological behavior. Figure 2 shows that the limiting low-shear viscosity of S. calva gel is higher in comparison with S. medusula gel. This is due to a higher concentration of the rheologic modifier (Carbopol ® Ultrez 21) in the S. calva formulation (Section 4.7). It is observed that the shear viscosity of the formulations decreases with increasing shear rate after a sufficiently large rate is attained. The descending part of the flow curve confirmed that both formulations exhibited non-Newtonian viscosity, i.e., pseudoplastic behavior [31].

Rheology and Extensibility of Sloanea-Extract-Based Gels
Viscosity is frequently employed to analyze the characteristics of a fo ensure optimal distribution. It also enables formulators to enhance the effica care products because this can be adjusted according to specific application n In this study, the viscosity (Pa·s) of the gels was analyzed as a functio rate (1/s) to monitor the stability of the formulation and define its rheolog Figure 2 shows that the limiting low-shear viscosity of S. calva gel is higher with S. medusula gel. This is due to a higher concentration of the rheologic bopol ® Ultrez 21) in the S. calva formulation (Section 4.7). It is observed viscosity of the formulations decreases with increasing shear rate after a su rate is attained. The descending part of the flow curve confirmed that both exhibited non-Newtonian viscosity, i.e., pseudoplastic behavior [31]. The measurement of the extensibility area provides information abo mation threshold of the formulations [32]. The extensibility area was measu ing the increase in the surface of the gels under progressively increasing pre g, 1.1624 g, and 4.2465 g) at 1 min intervals. This analysis resulted in the de extensibility area values, which provide valuable information about the cap dusula-and S. calva-based gels to stretch over the skin surface without exper age. Under these conditions, the S. medusula-based gel exhibited an extens 10.78 ± 2.5 cm 2 , showing a comparable elasticity or flexibility to the S. calva-b an extensibility area of 10.56 ± 3.1 cm 2 .

Efficacy Studies
Evaluation of the Antioxidant Activity of Extract-Based Gels by DPPH Assa The results of antioxidant activity expressed as DPPH radical scavengin and TEAC of gels based on S. medusula and S. calva extracts, respectively, The measurement of the extensibility area provides information about the deformation threshold of the formulations [32]. The extensibility area was measured by observing the increase in the surface of the gels under progressively increasing pressures (0.5958 g, 1.1624 g, and 4.2465 g) at 1 min intervals. This analysis resulted in the determination of extensibility area values, which provide valuable information about the capacity of S. medusulaand S. calva-based gels to stretch over the skin surface without experiencing breakage. Under these conditions, the S. medusula-based gel exhibited an extensibility area of 10.78 ± 2.5 cm 2 , showing a comparable elasticity or flexibility to the S. calva-based gel with an extensibility area of 10.56 ± 3.1 cm 2 .

Efficacy Studies Evaluation of the Antioxidant Activity of Extract-Based Gels by DPPH Assay
The results of antioxidant activity expressed as DPPH radical scavenging activity (%) and TEAC of gels based on S. medusula and S. calva extracts, respectively, are shown in Table 6. Antioxidant activity values were obtained at a concentration of 0.3 mg/mL. It is observed that the S. medusula gel exhibits the highest antioxidant capacity (54.5%, 22.9 mM Eq. Trolox/100 g of sample weight) compared to the S. calva-extract-based gel (53%, 20.3 mM Eq. Trolox/100 g sample weight). Table 6. Results of DPPH assays of extract-based gels. The results of the determination of SPF for the two gels based on Sloanea extracts (0.3%) are shown in Table 7. To evaluate the photoprotective potential of the two formulations, a 1:1 ratio of gel and ethanol was prepared. The photoprotection indices obtained were FPS 60.0 and FPS 57.3 for the gels based on S. medusula and S. calva, respectively. The same methodology was used to evaluate the effectiveness of commercial sunscreen. Table 7. Results of determination of sun protective factor of Sloanea-extract-based gels.

Formulation
SPF a MEAN ± SEM (n = 3) Sun protective factor. b Maximum UV protection. c High UV protection. d Good UV protection. f A commercial product that is marketed as having SPF 50. Results were expressed as the mean ± standard error of the mean from three independent assays.
In addition to SPF, the UVA/UVB ratio, the transmission of erythema (%), and the transmission of pigmentation (%) were calculated following the criteria used in Section 2.1.3. Results are shown in Supplementary Table S2.
Evaluation of the Anti-C. albicans Activity of Extract-Based Gels Two final concentrations (0.15% and 0.3%) of each gel were evaluated following the same protocol used to determine the MIC of Sloanea extracts against Candida species using 96-wheel plates. The experimental design is shown in Supplementary Figure S9. Then, three independent assays of colony-forming units were developed (Figure 3). The results of the inhibitory activity against C. albicans ATCC 10231 were expressed as the geometric mean of colony-forming units per milliliter (GM-CFU/mL) and percentage of yeast growth inhibition ( Table 8).
The gel based on S. medusula showed the highest activity, with a 98% inhibition of C. albicans growth at a final concentration of 0.15%. Another notable result was obtained with the gel based on S. calva, which achieved a 76% inhibition of yeast growth at a concentration of 0.3%. Figure 4 corresponds to the results obtained in one of the assays developed for the determination of the percentage of inhibition of yeast growth in the formulations. Each white spot corresponds to a colony or colony-forming unit.  67,824 0 a Data are the geometric mean of colony-forming units per milliliter (n = 3). b The base gel was used as a positive control (growth control of C. albicans). The comparisons of results between the different groups were performed statistically using the paired t-test. * Significant difference in inhibitory activity when compared to the control group (p < 0.05).
The gel based on S. medusula showed the highest activity, with a 98% inhibition of C. albicans growth at a final concentration of 0.15%. Another notable result was obtained with the gel based on S. calva, which achieved a 76% inhibition of yeast growth at a concentration of 0.3%. Figure 4 corresponds to the results obtained in one of the assays developed for the determination of the percentage of inhibition of yeast growth in the formulations. Each white spot corresponds to a colony or colony-forming unit.

Statistical Analysis
The results were expressed as the mean ± standard error of the mean. The experimental data were collected at least in triplicate for each activity. The comparisons of results between the different groups were performed statistically using the paired t-test. The statistical significance was set at p < 0.05. GraphPad Prism 8.0 (GraphPad Software, San

Statistical Analysis
The results were expressed as the mean ± standard error of the mean. The experimental data were collected at least in triplicate for each activity. The comparisons of results between the different groups were performed statistically using the paired t-test. The statistical significance was set at p < 0.05. GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA) was used to show the flow behavior of Sloanea-extract-based gels in rheology analysis.

Discussion
Sloanea is a genus of shrubs and trees in the family Elaeocarpaceae, mainly distributed in tropical regions [33], which comprises approximately 150 species [34]. Sloanea chocoana is an endemic species of the Chocó Department in Colombia, which is found in the central rainforest of this region [35].
Sloanea calva Palacios-Duque & Fern. Alonso sp. nov. ( Figure 5) is a tree that grows from 0 to 135 m.a.s.l, ranging from 15 to 40 (52) m in height, and can be found in both primary and secondary forests in the Pacific and Central regions of Chocó. It is known as "Pulgo" in the municipality of Nuquí (Pacific coast) and as "Táparo" in the village of Salero, municipality of Unión Panamericana [36]. S. medusula K. Schum. & Pittier ( Figure 6) is a tree that can reach heights between 12 and 35 m and is typically found at elevations ranging from 0 to 500 m.a.s.l. It has a broad distribution in the lowland areas of both the Caribbean and Pacific slopes, ranging from the central Pacific to the Osa Peninsula. In Nicaragua and Costa Rica, it is known as "Alma negra", "Peine de mico", and "Mano de león" [37]. S. medusula K. Schum. & Pittier ( Figure 6) is a tree that can reach heights between 12 and 35 m and is typically found at elevations ranging from 0 to 500 m.a.s.l. It has a broad distribution in the lowland areas of both the Caribbean and Pacific slopes, ranging from the central Pacific to the Osa Peninsula. In Nicaragua and Costa Rica, it is known as "Alma negra", "Peine de mico", and "Mano de león" [37]. Some species of Sloanea have traditionally been used in medicinal practices to treat ailments related to infectious and inflammatory processes [12], and their wood is often used to make axe handles, as well as for fuel or firewood [34].
The compounds identified in Sloanea species have shown interesting biological properties, which include the antifungal and antiplasmodial activity exhibited by some galloylquinic acid derivatives [13,38], and cytotoxic activity against human cancer cells, including breast (MCF-7), lung (H-460), and central nervous system (SF-268) exhibited by cucurbitacin analogs [14]. The chemical structures of some polyphenol and triterpenoid compounds identified in Sloanea species are shown in Figure 7. Some species of Sloanea have traditionally been used in medicinal practices to treat ailments related to infectious and inflammatory processes [12], and their wood is often used to make axe handles, as well as for fuel or firewood [34].
The compounds identified in Sloanea species have shown interesting biological properties, which include the antifungal and antiplasmodial activity exhibited by some gal-loylquinic acid derivatives [13,38], and cytotoxic activity against human cancer cells, including breast (MCF-7), lung (H-460), and central nervous system (SF-268) exhibited by cucurbitacin analogs [14]. The chemical structures of some polyphenol and triterpenoid compounds identified in Sloanea species are shown in Figure 7. In our study, the HPLC-MS analysis of the two extracts led to the tentative identification of seven compounds, including glycolic acid 4-hydroxy-3,5-di-t-butylbenzyl ester (identified in both Sloanea species), α-sorinin and two hydrolyzable tannins (granatin B and geraniin) for S. medusula, and three pentacyclic triterpenoids (uralenic acid, asiatic acid, and asiatic acid triacetate) for S. calva.
The peak at TR 9.882 and m/z 293 was identified as the ion [M-H] of glycolic acid 4hydroxy-3,5-di-t-butylbenzyl ester (C17H26O4). The fragment ion analysis and neutral losses in the MS-MS spectrum suggest the loss of a glycolic acid unit (76 Da) and 2-methylpropane unit (58 Da) resulting in peaks at m/z 219 (C15H23O) and m/z 236 (C13H17O4) (Supplementary Figure S2). According to our review, this compound did not yield results from previous studies of fragment ion analysis in the ESI-MS 2 spectrum.
The peak at TR 3.4207 and m/z 539.123 was identified as the ion [M-H] of α-sorinin (C24H28O14), a compound related to the antioxidant activity observed in naphthalenic compounds from Rhamnus nakaharai [39]. The fragment ion analysis suggests the loss of a glucose unit and deoxyribopyranose unit for a total neutral loss of 314 Da, resulting in the peak at m/z 227 (C13H7O4) (Supplementary Figure S3). The molecular ion is observed at m/z 313 (C11H21O10) as a result of a neutral loss of the aglycone unit (226 Da). Finally, a total neutral loss of 152 Da corresponding to the deoxyribopyranose unit and water unit, led to fragment ion at m/z 387. This compound did not yield results from previous studies of fragment ion analysis in the ESI-MS 2 spectrum.
The peak eluted at TR 3.2289 and m/z 476.033, integrated for two compounds. This was identified as the [M-H] ion of ellagitannins corresponding to the formula C41H28O27 (tentative annotations: geraniin and granatin B). Ellagitannins are high molecular weight plant polyphenols found in woody and non-woody plants, which perform plant defense functions and are natural antioxidants that have demonstrated benefits for human and animal health due to their antimicrobial and antiparasitic properties [40]. The fragment ion analysis suggests the neutral loss of 782 Da and 651 Da, resulting in two peaks at m/z 169 (C7H5O5) and m/z 300 (C14H5O8) that correspond to the galloyl group (molecular ion) and a possible ellagic acid ion (Supplementary Figure S4 and S5), respectively. In the literature, ESI-MS analysis of geraniin (positive ion mode) has shown fragment ions at m/z 951, 554, 446, and 247 [41], and granatin B (negative ions mode) at m/z 951, 783, 605, and 300 [42]. In our study, the HPLC-MS analysis of the two extracts led to the tentative identification of seven compounds, including glycolic acid 4-hydroxy-3,5-di-t-butylbenzyl ester (identified in both Sloanea species), α-sorinin and two hydrolyzable tannins (granatin B and geraniin) for S. medusula, and three pentacyclic triterpenoids (uralenic acid, asiatic acid, and asiatic acid triacetate) for S. calva.
The peak at T R 8.044 and m/z 471.346886 was tentatively identified as the ion [M+H] of Uralenic acid or glycyrrhetinic acid (C 30 H 46 O 4 ) (Supplementary Figure S6), a pentacyclic triterpenoid of oleanane-skeleton with remarkable antioxidant and antitumoral effects [43]. The fragments corresponding to ring breakage showed peaks at m/z 205 (C 15 [47].
In general, identified compounds in HPLC-MS analysis have exhibited properties that benefit skin health, such as antimicrobial, antioxidant, anti-inflammatory, chemoprotective, cytotoxic, and wound-healing properties [49][50][51][52]. Therefore, these compounds may be responsible for the observed biological activities. These findings create an opportunity for bioprospecting and evaluating the potential use of extracts from other Sloanea species, active fractions, or compounds in the cosmetic and pharmaceutical industries.
Various studies have shown the potential of plant extracts in skincare product development, especially for cutaneous candidiasis treatment induced by C. albicans [53]. In addition, the protective effects against premature aging have been addressed, due mainly to the photoprotective and antioxidant properties of natural extracts [54]. Regarding formulations, gels have emerged as a promising alternative for skin infection treatments. Their use implies lower toxicity compared to oral or intravenous antifungals, excellent chemical stability, and enhanced penetration in infected areas, making them effective delivery agents for multiple compounds [55]. The benefits of plant-extract-based gels have been observed in emulgels loaded with clove/cinnamon oils, which are an alternative for treating C. albicans-associated denture stomatitis [56], and in hydrogels integrating lemongrass-loaded nanosponges showing an enhanced antifungal effect with reduced irritation [57].
Currently, the photoprotective properties of extracts [58], essential oils [29], and secondary metabolites [59] are being explored, and the antifungal activity of these natural extracts [60] and compounds [61] has highlighted the potential of Colombian flora for the development of topical formulations.
In this study, ethanolic extracts obtained from S. medusula and S. calva collected in Chocó, Colombia, exhibited antioxidant effects, with values that are considered satisfactory for natural extracts [62]. This activity plays a fundamental role in the photoprotective and antifungal properties observed in natural extracts [63,64]. Results obtained showed that the extracts and Sloanea-extract-based gels offer high and maximum SPF values. Moreover, a noteworthy reduction in values in vitro of percentages of transmission of erythema and pigmentation (Supplementary Table S2) suggests the ability of these extracts and formulations to protect the skin exposed to sunlight from redness and dark spots, respectively. The results obtained with the evaluated extracts exceed those reported for Holoptelea integrifolia extract (Ulmacea), a species that shares traditional uses similar to those reported for the Sloanea species, including malaria, rheumatism, common fever, as well as other conditions related to rickets, leprosy, leucoderma, among others [65].
Our results demonstrate that extracts from S. medusula and S. calva exhibit significant antifungal properties, particularly against the yeast C. albicans, and these properties are preserved in the gels. However, the disparity observed in the gels made with S. medusula is noteworthy, where a concentration of 0.15% of the extract showed a superior inhibitory effect compared to the highest concentration evaluated. Concerning that, phenomena have been described in the literature in different species of Candida exposed to echinocandins (an antifungal whose target is the enzyme B-1,3 glucan synthase, responsible for the synthesis of glucans in the fungal wall). This phenomenon is known as a paradoxical effect. It is caused by exposure to high concentrations of echinocandins, which induce stress adaptation pathways by increasing cell wall chitin in vitro in response to the depletion of wall glycans and contributing to fungal growth [66]. This growth makes the interpretation of in vitro susceptibility tests difficult. However, its relevance in vivo has not been proven. Although our results do not allow us to elucidate what is happening, the increase in the growth of C. albicans at high concentrations of the S. medusula extract and its inhibition at low concentrations could be related to a similar effect.
The gels obtained showed pseudoplastic behavior and extensibility areas that lead to a favorable dispersion during its topical application, which favors the creation of a uniform film on the skin surface, contributing to the efficacy of the formulations [67]. However, a decreased antifungal efficacy of the S. calva-based gel was observed, which could be mainly attributed to the composition of the polymer within the gel. The literature suggests that the active ingredients in the gel, such as crude extracts, can strongly interact through the -OH, -COOH, and C-O-C groups with the polymer via hydrogen bonding [68]. This interaction results in higher viscosity and reduced release capacity of the active compounds. Therefore, adjusting the polymer composition within the formulation may lead to improved efficacy.
Although it is not common for topical treatments for infections caused by C. albicans to cause skin pigmentation, some treatments can cause irritation or dryness (e.g., Gentian violet, Nystatin, Miconazole, Ketoconazole, and Clotrimazole) and ulcers (e.g., Gentian violet), making the skin more sensitive to sunlight and potentially leading to dark spots [69].
It is important to note that using a formulation with both SPF and anti-Candida albicans activity can be highly beneficial. Sunscreen helps protect the skin from UV damage, which can avoid skin irritation and pigmentation issues. Incorporating an anti-C. albicans agent into the formulation can help address the root cause of the infection and prevent further occurrences.
Today, cutaneous candidiasis caused by C. albicans has emerged as a critical health concern, demanding immediate attention and concerted efforts toward continuous research and development of therapeutic options. An enhanced prevalence of cutaneous candidiasis becomes a substantial burden for healthcare systems and public health in general [18]. Hence, prioritizing and investing in ongoing research becomes pivotal to mitigate the adverse impact of this disease and enhance the quality of life for affected individuals.
Future directions of this study will lead to the optimization of both gels to enhance their antifungal properties. The optimization of viscosity and rheological behavior of the gels should be accompanied by pH measurement, which should be ≤5.5 for topical formulations [70], to preserve the integrity of the skin barrier [71]. This should be followed by in vitro tolerance studies, evaluation of genomic integrity, and in vivo studies. Furthermore, it would be interesting to study the underlying mechanisms related to the inhibitory effect on the growth of C. albicans using the gel based on S. medusula extract.

Materials
All available reagents and solvents were obtained from commercial suppliers and used without further purification. These were acquired from Fisher Scientific (

Plant Material Collection and Extraction
The plants under study were collected in Quibdó, Chocó Department, Colombia For each specimen, the leaves were air-dried, followed by a grinding process using a ball mill. The ground plant material was macerated (no stirring) with 96% ethanol for 72 h. The solvent was filtered, and the residue was extracted two-fold. The resulting extracts were evaporated and dried using rotary evaporation until the crude extracts were obtained. The yield of each extract was 1.1% (S. calva) and 1.2% (S. medusula) relative to the fresh plant. The obtained extracts were labeled and stored at 4 • C until use.
The extraction was carried out using ethanol, as it is one of the most commonly used solvents in the traditional medicine practices of Chocó communities. This solvent facilitates the extraction of compounds with therapeutic properties of interest, such as flavonoids, tannins, phenolic compounds, and triterpenoids, among others [72].

HPLC-MS Analysis
The extracts were separately diluted in methanol. Each diluted sample was vigorously shaken to achieve homogenization. Subsequently, a filtering process was applied using a 0.22 µm pore size membrane, and the filtered samples were transferred to autosampler vials. The extract was separated and analyzed using a UHPLC (Dionex Ultimate 3000, Thermo Scientific, Waltham, MA, USA), coupled to an ESI q-QTOF (Impact 2, Bruker Daltonics), operated in positive and negative ion modes. Separation was carried out using a Luna C18 column, 4.6 × 150 mm, 3 µm particle size (Phenomenex, Torrance, CA, USA). The column temperature was maintained at 30 • C. The mobile phase consisted of water (A) and acetonitrile (B). The analysis started with 40:60 A: B, held for 5 min, followed by changing linearly up to 100% B in 5 min, then returned to 40:60 A: B in 5 min, and held until 25 min. The flow was 0.4 mL/min and the injection volume of 5 µL. The conditions for the mass detector were as follows: Capillary voltage +3.5 kV (negative ion) and +4.5 kV (positive ion), nitrogen gas temperature 220 • C, drying gas flow rate 8.0 L/min, and nebulizer gas pressure 1.8 Bar. MS/MS data acquisition mode was used to assist compound identification. The mass range in MS/MS experiments was set at m/z 50-1300. Global Natural Products Social Molecular Networking (GNPS; http://gnps.ucsd.edu accessed on 6 June 2023) was used for the analysis of MS/MS data [73].

DPPH Assay
The study assessed the hydrogen atom donating ability of plant extracts and gels by measuring the decolorization of a methanol solution of DPPH, which changes from violet/purple to shades of yellow in the presence of antioxidants [74]. The DPPH radical scavenging assay was performed following the method reported in the literature [75], with some modifications. Briefly, a solution of DPPH 0.125 mM was prepared by dissolving 0.00197 g of the DPPH radical in 15 mL methanol, and the volume was adjusted with acetate buffer until the absorbance of the working solution was 1.0 ± 0.2 at 517 nm. The standard solution was prepared by dissolving 2 mg of Trolox (97% purity) in 10 mL of 80% methanol. The solution obtained had a deep purple color and was left in the refrigerator for 2 h for the absorbance to be stabilized. Then, a curve of Trolox standard solution was prepared in a concentration range from 0.08 mM to 2 mM. These solutions were vortexed and left in darkness and their absorbance was measured using a Varioskan™ LUX Multimode Microplate Reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA) at λ max of 517 nm. The calibration curve of absorbance (y) versus concentration (x) of DPPH is expressed by the following equation (y = −0.16x + 0.4792, R 2 = 0.9973). Subsequently, 0.05 mL of extract solution (0.7 mg/mL) or gel (0.3 mg/mL) is added to 1.95 mL of the DPPH radical working solution. The mixture is carefully homogenized and kept in the dark for 30 min. Finally, 200 µL per triplicate is dispensed into a 96-well plate and the absorbance is measured at 570 nm. For the antioxidant assay, methanol was used as the blank.
The percentage of DPPH radical scavenging activity was calculated by the following equation: where A 0 is the absorbance of the control, and A 1 is the absorbance of the Sloanea extracts/extracts-based gels. Then, the results expressed as TEAC (Trolox equivalent antioxidant capacity) (µmolar/100 g sample weight) were calculated from a calibration curve obtained by linear regression, following the equation: µM Eq. Trolox 100 g sample weight = M * FD * V m * 100 (2) where M is the molar concentration obtained from the calibration curve, FD is the dilution factor used to prepare the sample, V is the volume in liters in which the sample was initially prepared, and m is the weight in grams of the initial sample.

Evaluation of Sun Protection Factor of Sloanea Extracts and Sloanea-Extract-Based Gels
The SPF was determined by measuring the absorbance values between wavelengths of 290 to 320 nm (UVB spectrum) using an optical path of 5.0 cm in a Varioskan ™ LUX Multimode Microplate Reader (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The calculated SPF value was then applied to the Mansur equation [76]: In this equation, CF represents the correction factor (equal to 10), while EE(λ) × I(λ) are constants at each wavelength (Table 9) obtained from the correlation between the erythemal effect (EE) of radiation of wavelength λ and the solar intensity at the same wavelength (I) [77]. Additionally, Abs refers to the absorbance of the solution at wavelength λ [78]. The CF value was determined so that a standard sunscreen formulation containing 8% homosalate would have an SPF value of 4, measured by UV spectrometry [79]. SPF values between 2-15 indicate low protection against UV rays, values between 15-30 indicate moderate protection, values between 30-50 indicate high protection, and values greater than 50 indicate maximum protection against UV rays [29].  [80], it was mandatory that the MIC values remained within the accepted range, i.e., MIC values of 0.125 µg/mL and 0.630 µg/mL for ITC and AMB, respectively [81]. All yeasts were cultured on Sabouraud Dextrose Agar for 24 h at 35 • C.

Antifungal Susceptibility Testing
The antifungal susceptibility testing was performed according to the CLSI standard M27, 4th Edition [80]. Some modifications were applied to evaluate the antifungal effects of extracts. Briefly, stock solutions of extracts were prepared at 512 µg/mL, and an inoculum of 2.5 × 10 3 CFU/mL of each yeast was prepared in RPMI 1640-MOPS. Then, 100 µL of each stock solution of extracts were added to 96-well microdilution plates to finally evaluate 10 two-fold dilutions ranging from 0.5 to 256 µg/mL, followed by 100 µL of each yeast inoculum (evaluated concentration of the inoculum was 1.25 × 10 3 CFU/mL). The microdilution plates were again incubated at 35 • C for 24 h. The MICs were visually determined as the lowest concentration that produced visual inhibition compared to the growth control using a manual mirror viewer. The assays were performed at least three times in duplicate on different days, and the results were expressed as GM-MIC.
The antifungal susceptibility testing described to evaluate the antifungal effects of extracts was used to evaluate the susceptibility of C. albicans against the Sloanea-extract-based gels at two final concentrations (0.15% and 0.3%), including the gel base as a growth control (Supplementary Figure S9). To obtain results of the antifungal activity of the gels at a final concentration of 0.3%, formulations were prepared by doubling the concentration of the extract (i.e., 0.6% w/w) while maintaining the same concentrations of the other components in the formulation. An inoculum of 2.5 × 10 3 CFU/mL of C. albicans was prepared in RPMI 1640-MOPS (2X) to evaluate the antifungal effects of the base gel and Sloanea-based gels. Gel (100 µL) was added to 96-well microdilution plates, followed by 100 µL of inoculum (the evaluated final concentration of the inoculum was 1.25 × 10 3 CFU/mL). After 24 h of incubation at 35 • C, the colony-forming unit assay was performed to determine the CFU/mL, and the results were expressed as GM-CFU/mL and yeast growth inhibition (%). The CFU assay involved the preparation of 1:10, 1:100, and 1:1000 dilutions. To prepare 1:10 dilutions, one well per concentration was randomly chosen (Supplementary Figure  S9). A volume of 10 µL was taken from the well and diluted in sterile deionized water (90 µL). Subsequently, 1:100 and 1:1000 dilutions were prepared. Once the dilutions were prepared, a volume of 10 µL was taken, which was transferred to the center of each plate and spread in the solid agar medium (Sabouraud dextrose agar) with the help of 4-5 glass beads for the purpose of allowing viable cells to form colonies under specific growth conditions. The beads were removed, and the plates were subjected to an incubation period at 35 • C. After 24 h, the colonies were counted, and the CFU/mL was calculated based on the 1:10 dilution factor.

Preparation of Sloanea-Extract-Based Gels, Rheology, and Extensibility
To prepare both gels, Sloanea extract was first dissolved in deionized water. This solution was then added to a mixture containing carbopol ® ultrez 21 polymers, sodium nipagin, triethanolamine, glycerine, and 100% deionized water q.s.p. (Table 10), which was prepared at 40 • C. The mixture was thoroughly dissolved. Additionally, a blank formulation (negative control) was prepared under the same conditions, excluding the incorporation of the extract. During the preparation of the base formulation and optimization of the final formulations, differences were observed in the behavior of the base gel when it came into contact with the plant extract. First, the rheology modifier (Carbopol) in the base gel was adjusted until the standardization of the S. medusula-based gel. Then, to standardize the gel based on S. calva, it was necessary to increase the concentration of Carbopol until a formulation that offered rheological properties similar to those obtained with the first extract was achieved. These tests resulted in the concentrations shown in Table 9. The physicochemical behavior of the gels against evaluated natural extracts would be an interesting topic to address in future research.
The organoleptic properties of the gels based on Sloanea extracts were assessed by two individuals at different times. Images of gels were acquired using a stereomicroscope with a 2× objective (Zivot).
Rheological analysis was performed using an MCR92 rheometer (Anton-Paar ® , Graz, Austria). The viscosity of the gels was run with 50 mm cone-and-plate to 25 ± 0.5 • C. Samples of the two gels were stored at room temperature. Apparent viscosity and rheological behavior were determined at 28 days. The graph of flow behavior was generated using GraphPad Prism 8.0.
The extensibility area of the gels was determined at a room temperature of 25 ± 0.5 • C using the methodology of Pérez-Bueno et al. [32], with minor modifications. Briefly, a known amount of the sample was placed at the center of a flat glass surface. Subsequently, another flat glass plate with a known weight (average: 0.5958 g) was placed on top of the sample. After waiting for 1 min, the extended radius of the sample was measured. The same measurement was repeated after adding two additional flat plates with known weights (average values: 1.1624 g and 4.2465 g, respectively). The measurements were performed in triplicate. Based on the three data points, a curve of radius vs. weight was plotted, and the slope expressed in cm/g was determined.
The slope of S. medusula gel was 2.917 cm/g for an average sample weight of 0.635 g, and the slope of S. calva gel was 2.095 cm/g for an average sample weight of 0.875 g.
The average radius was then calculated through the equation: Average radius (cm) = slope × average weight (4) Finally, the extensibility area expressed in cm 2 was calculated using the following equation: Extensibility area (cm 2 ) = π(average radius) 2 (5)

Conclusions
This study constitutes the first report on the chemical composition and biological activities of the ethanolic extracts obtained from S. medusula and S. calva leaves.
Chemical composition was studied using the HPLC-MS method. Two hydrolyzable tannins (granatin B and geraniin) were tentatively identified in S. medusula extract, while three pentacyclic triterpenoids (uralenic acid, asiatic acid, and asiatic acid triacetate) were tentatively identified in S. calva extract. These compounds are widely recognized as antioxidant, anticancer, and antimicrobial agents, suggesting a relation with the observed biological activities.
Our results indicated that both extracts possess high antioxidant and photoprotective activity. In addition, promising antifungal activity was observed against different Candida species, although the susceptibility of the tested strains varied. MIC values of 2 µg/mL were obtained against C. albicans, a yeast species associated with cutaneous candidiasis. In summary, these results suggest the beneficial effects of S. medusula and S. calva extracts on the skin, and further research is recommended to determine the effects of these extracts on human health.
The gel based on S. medusula evaluated at a final concentration of 0.15%, offers suitable characteristics as an anti-Candida albicans agent. This makes it an attractive formulation and could constitute a promising alternative in cutaneous candidiasis treatment.
Future directions for this work include the in vitro tolerance studies, evaluating genomic integrity, and performing in vivo studies. Additionally, the study of underlying mechanisms behind the inhibitory effect on C. albicans growth using the gel containing S. medusula extract and the physicochemical behavior of gel based on S. calva extract are of particular interest.  Table S1: Proposed fragments and proposed neutral losses for the tentative annotations identified in ethanolic extracts from S. medusula and S. calva; Figure S2: 2D chemical structure (drawn in black color), proposed fragments (drawn in blue color), proposed neutral losses (drawn in red color), and MS-MS spectrum for the tentative annotation glycolic acid 4-hydroxy-3,5-di-t-butylbenzyl ester; Figure S3: 2D chemical structure (drawn in black color), proposed fragments (drawn in blue color), proposed neutral losses (drawn in red color), and MS-MS spectrum for the tentative annotation α-sorinin; Figure S4: 2D chemical structure (drawn in black color), proposed fragments (drawn in blue color), proposed neutral losses (drawn in red color), and MS-MS spectrum for the tentative annotation geraniin; Figure S5: 2D chemical structure (drawn in black color), proposed fragments (drawn in blue color), proposed neutral losses (drawn in red color), and MS-MS spectrum for the tentative annotation granatin B; Figure S6: 2D chemical structure (drawn in black color), proposed fragments (drawn in blue color), proposed neutral losses (drawn in red color), and MS-MS spectrum for the tentative annotation uralenic acid; Figure S7: 2D chemical structure (drawn in black color), proposed fragments (drawn in blue color), proposed neutral losses (drawn in red color), and MS-MS spectrum for the tentative annotation asiatic acid; Figure S8: 2D chemical structure (drawn in black color), proposed fragments (drawn in blue color), proposed neutral losses (drawn in red color), and MS-MS spectrum for the tentative annotation asiatic acid triacetate; Table S2: In vitro photoprotective activity measurement of ethanolic extracts and extract-based gels of Sloanea medusula and S. calva; Figure S9: Experimental design for evaluating the anti-Candida albicans activity of gels based on S. medusula and S. calva.