Psidium cattleyanum Sabine as a Source of Bioactive Compounds for Skin Disorders

TL;DR

This study explores the potential of strawberry guava leaves from Cabo Verde as a natural source of compounds beneficial for skin health, including antioxidant, anti-aging, and antimicrobial properties.

Contribution

The study identifies and characterizes the bioactive potential of Psidium cattleyanum leaf extract for dermatological applications, including its formulation into a stable topical cream.

Findings

The leaf extract showed strong antioxidant activity and inhibited enzymes linked to skin aging.

It exhibited antimicrobial effects against common skin pathogens like Staphylococcus aureus and Cutibacterium acnes.

The extract was successfully formulated into a stable topical cream suitable for skin care.

Abstract

Psidium cattleyanum Sabine (strawberry guava, araçá) is an ethnomedicinal plant with reputed health benefits; however, its potential for treating skin disorders remains underexplored. This study aimed to characterize the phytochemical profile of P. cattleyanum leaves from Cabo Verde and evaluate their bioactivity relevant to skin health. Phytochemical analysis was performed using high-performance liquid chromatography-mass spectrometry (LC-MS) and spectrophotometric assays. Key biological activities were assessed in vitro, including antioxidant capacity (free radical scavenging assays), anti-aging enzyme inhibition (collagenase, elastase, and tyrosinase), and antibacterial activity against skin pathogens (agar diffusion, minimum inhibitory concentration, and combination studies with standard antibiotics). Cytotoxicity was evaluated using Vero cells (MTT assay). Additionally, a topical cream containing the leaf extract was formulated and subjected to physicochemical stability and sensory testing. LC-MS revealed a rich polyphenolic composition in the leaf extract, including abundant phenolic acids (gallic and ellagic acid derivatives) and flavonoid glycosides. The extract exhibited a high total phenolic content and strong antioxidant activity in DPPH/ABTS assays. It showed potent inhibition of collagenase, elastase, and tyrosinase, indicating an anti-aging effect against wrinkle formation and hyperpigmentation. The extract also demonstrated broad antimicrobial efficacy against skin-associated bacteria, such as Staphylococcus aureus and Cutibacterium acnes, with no antagonism and partial synergism observed when combined with certain antibiotics. The P. cattleyanum extract was successfully incorporated into a cream formulation that remained physically and chemically stable (no phase separation, consistent droplet size, and pH) over 90 days, with good homogeneity and acceptable sensory characteristics (neutral odor, smooth texture, and good spreadability). P. cattleyanum leaves from Cabo Verde are a rich source of bioactive compounds with multifunctional dermatological benefits. This study demonstrates that the P. cattleyanum leaf extract exhibits significant antioxidant, antimicrobial, and anti-aging activities in vitro, supporting its potential use as a natural ingredient for skin care.

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1. Introduction

Psidium cattleyanum Sabine, commonly known as araçá or goiavinha, is a small fruiting tree in the Myrtaceae family native to Brazil and is widely distributed in tropical regions worldwide [1]. It has been introduced to various locales (including West African islands such as Cabo Verde), where it is valued for its edible fruits [2]. The red or yellow berry-like fruits have a sweet, aromatic flavor and are exceptionally rich in vitamin C, containing about 3–4 times more vitamin C than citrus fruits [1]. In local Cabo Verdean parlance, the plant is referred to as araçá or “goiavinha,” and although formal ethnopharmacological documentation in Cabo Verde is limited, the species is presumed to be utilized similarly to its Brazilian counterpart [2]. In Brazilian folk medicine, P. cattleyanum is used to treat various ailments. For example, decoctions of the leaves are traditionally used to combat diarrhea, digestive disorders, and abdominal pain, and the plant has also been used as a diuretic and to control blood pressure and hemorrhage [3]. Such ethnomedicinal uses suggest the presence of bioactive phytochemicals, and scientific studies in recent years have begun to validate many of these traditional claims. P. cattleyanum leaves are reported to possess notable anti-diarrheal activity in folk practice, although until recently no thorough scientific investigation had confirmed this claim [4]. Moreover, related Psidium species (especially the common guava, P. guajava) are well known in traditional medicine for their antioxidant, antimicrobial, antispasmodic, and anti-inflammatory effects [5], indicating that P. cattleyanum may have a similar pharmacological potential.

Modern phytochemical research has revealed that P. cattleyanum is rich in secondary metabolites, particularly polyphenols and terpenoids, which are likely to underlie its biological activities. The fruits and leaves of P. cattleyanum contain abundant phenolic compounds, including flavonoids and tannins, which have been linked to a broad spectrum of bioactivities, including antioxidant, antimicrobial, antiproliferative, antidiabetic, and anti-inflammatory effects [1,6]. For instance, ripe fruits (araçá) have been noted for their high antioxidant content and are considered a potential source of natural antioxidants for food and health applications [7]. The leaves have demonstrated antimicrobial and anti-inflammatory properties in vitro, supporting their folkloric use in wound healing and diarrhea remedies [8,9]. The rising interest in P. cattleyanum also stems from its potential applications in the pharmaceutical, nutraceutical, and cosmetic industries. Preliminary reports have highlighted P. cattleyanum leaf extracts as possible natural preservatives and skin-protective agents, and patents have been filed for skincare products (lotions, soaps) containing P. cattleyanum extracts, underscoring the cosmetic relevance of this species. Despite this promise, significant gaps remain in our knowledge; in particular, the detailed phytochemical profile of P. cattleyanum from regions such as Cabo Verde has not been studied, nor have its dermatological benefits been rigorously evaluated. Geographical variation is known to affect the phytochemistry of medicinal plants; indeed, the chemical composition of P. cattleyanum can shift with differing climates and locales. Investigating the Cabo Verde population of P. cattleyanum is scientifically relevant because this archipelago’s unique environmental conditions (soils, semi-arid climate, and insular ecology) may yield a distinct chemotype with potentially novel or enhanced bioactivities. Moreover, since formal ethnopharmacological documentation in Cabo Verde is limited, studying a locally grown P. cattleyanum provides new insights into its medicinal potential in a West African island context.

To address these gaps, the present study was designed to provide a comprehensive analysis of P. cattleyanum leaves from Cabo Verde, integrating chemical characterization with biological assays relevant to skin health. We performed extensive LC-MS-based phytochemical profiling to identify key constituents and investigated the extract’s antioxidant capacity, antimicrobial effects, and in vitro anti-aging activities (including inhibition of collagenase, elastase, and tyrosinase, which are associated with skin aging and hyperpigmentation). To our knowledge, this is the first study to report these skin-targeted biological activities of P. cattleyanum from Cabo Verde. By linking the ethnopharmacological background of P. cattleyanum with modern analytical and bioassay techniques, we aim to better understand the value of this plant as a source of bioactive compounds for the management of skin disorders. The findings of this study not only shed light on the phytochemical richness of P. cattleyanum in a new geographic context but also justify its traditional uses and potential incorporation into therapeutic or cosmetic applications targeting skin ailments.

2. Results and Discussion

This study provides novel insights into the chemical composition and bioactivity of P. cattleyanum leaves from Cabo Verde, revealing a complex array of polyphenols with significant dermatological potential.

2.1. Phytochemical Profile of P. cattleyanum Leaf Extract

Phytochemical analysis confirmed that Cabo Verde P. cattleyanum is exceptionally rich in phenolic constituents. The total phenolic content (TPC) of the P. cattleyanum leaf extract was measured using the Folin-Ciocalteu reagent, and the results were expressed as gallic acid equivalents (GAE) per gram of dry extract (DE). The total phenolic content of the leaf extract was high (449.38 ± 1.31 mg GAE/g dry extract) (Table 1), consistent with previous reports that P. cattleyanum accumulates a high abundance of polyphenols. For example, Biegelmeyer et al. (2011) reported that the red strawberry guava fruit extract contained 501.33 ± 0.02 mg GAE/100 g dry weight, whereas the yellow strawberry guava fruit extract contained 292.03 ± 0.03 mg GAE/100 g dry weight of total polyphenols [10]. A lower total polyphenol content was detected in P. cattleyanum fruits obtained in Cairo, Egypt (77.71 ± 1.59 mg GAE/g) [6]. Moreover, Dacoreggio et al. [11] evaluated the total phenolic content of two extracts obtained from P. cattleyanum leaves collected during summer and winter in Brazil. In leaves harvested in summer, the polyphenol content ranged from 101 to 121 mg GAE/g DE, whereas in those collected in winter, it ranged from 123 to 144 mg GAE/g DE [11].

The total phenolic acid content (TPAC) in the P. cattleyanum leaf extract was 21.04 ± 0.85 mg CAE/g of the dry extract.

The total flavonoid content (TFC) of the P. cattleyanum leaf extract was determined using the colorimetric method described previously [12]. The data are expressed as quercetin equivalents (QE) per gram of dry extract (DE). The results showed that the total flavonoid content in the extract was 1.51 ± 0.09 mg QE/g dry extract. The results obtained in our study were better than those reported for extracts derived from the fruits of P. cattleyanum and P. cattleyanum var. lucidum Hort. collected in Brasil—100.20 ± 0.0716 and 35.12 ± 0.1270 mg/100 g of quercetin of d. w., respectively [10].

Chromatographic analysis allowed the detection of secondary metabolites in the extract. In total, 27 compounds were characterized based on the observed UV-Vis and MS spectra. Wherever possible, comparisons with chemical standards or suitable literature were made. Detailed data are provided in Figure 1 and corresponding Table 2.

The extract was found to be a source of various polyphenolic compounds. Two major groups of phytochemicals were detected. Compounds 2–7, 9–13 and 16 were classified as ellagitannins based on the observed UV maximum at approximately 270 nm, which is characteristic of the gallic acid chromophore. Compound 1, gallic acid, was also present in the extract, with an m/z ion at 169 in the negative mode spectrum. Compounds 3 and 5 with m/z of 933 were identified as isomeric monomeric ellagitannins vescalagin and castalagin, the latter of which was previously confirmed in Psidium [4]. Additionally, two other monomeric compounds belonging to the same class were detected and identified as telimagrandin I (4) with m/z of 785 and pedunculagin (7) with m/z of 783 [14]. Other constituents belonging to the ellagitannin class were represented by both dimeric and monomeric compounds, but their structures could not be fully elucidated due to lack of standards and definitive spectroscopic data.

The second most abundant group of compounds present in the PC extract were flavonoids (19–25 and 27). Compounds 19 to 23 were identified as quercetin glycosides based on the presence of an aglycone ion in the MS/MS spectrum at m/z 301 and strong maxima at approximately 255 and 350 nm in the UV detection. Most of these constituents have been previously reported in Psidium, including quercetin 3-O-galactoside (19), quercetin 3-O-glucuronide (20), two isomeric quercetin 3-O-arabinosides (21 and 22), and quercetin 3-O-rhamnoside (23) [15,16]. Additionally, three kaempferol glycosides (24, 25, and 27) were detected based on the presence of an aglycone moiety at m/z 285 in the MS spectrum and UV-Vis maxima at approximately 260 and 345 nm.

The presence of ellagic acid (18) with a major ion at m/z 301 and a characteristic UV-Vis spectrum was detected in the sample. This compound can be considered both a precursor for ellagitannins or their degradation product due to the extraction process. The occurrence of vanillic acid O-hexoside (26) was also confirmed based on a previous report [16]. Interestingly, one flavan-3-ol derivative (15) was detected as a minor compound due to the maximum observed at 280 nm and the presence of catechin or epicatechin moiety in the MS/MS fragmentation at m/z 289. This indicates that PC (P. cattleyanum leaf extract) is a rich source of ellagitannins rather than condensed procyanidin derivatives.

Our findings corroborate and extend such reports by demonstrating that P. cattleyanum from Cabo Verde shares a similar polyphenolic “fingerprint” with plants from other regions. Interestingly, Beltrame et al. (2022) previously highlighted three flavonols—hyperoside (quercetin-3-O-galactoside), miquelianin (quercetin-3-O-glucuronide), and quercitrin (quercetin-3-O-rhamnoside)—as key markers in P. cattleyanum leaves [9]. We detected compounds consistent with flavonol glycosides in our LC-MS data, which were among the predominant peaks in the chromatographic profile. The concurrence of our results with those of Beltrame et al. [9] and El-Deeb et al. [4] indicates that P. cattleyanum leaves universally contain a core set of bioactive flavonoids and ellagitannins, although environmental or geographical factors might influence their relative abundance. In the context of Cabo Verde, this is the first detailed phytochemical profiling of P. cattleyanum, revealing a rich secondary metabolite composition that helps explain the plant’s versatile traditional uses.

Moreover, similar phytochemical profiles have been observed in P. cattleyanum leaf extracts prepared with different solvents, including ethanol-based extracts. For instance, González-Silva et al. (2022) identified numerous phenolic acids (gallic, protocatechuic, chlorogenic, caffeic, p-coumaric, trans-cinnamic, 4-hydroxybenzoic, and syringic acids) as well as the flavonol kaempferol in P. cattleyanum leaves using an ultrasound-assisted hydroalcoholic extraction [8]. Likewise, Brighenti et al. reported that an aqueous leaf extract contained at least three flavonoids (kaempferol, quercetin, and the anthocyanin cyanidin) and ellagic acid, reflecting a polyphenol-rich composition comparable to our findings [17]. In a recent study on P. cattleyanum leaves cultivated in Egypt, El-Deeb et al. (2024) found that gallic acid, ellagic acid, catechin, and multiple quercetin derivatives (e.g., hyperoside, isoquercitrin, quercitrin) were the dominant constituents—a profile consistent with reports from Brazil, Korea, and Mexico [6]. These comparisons underscore that regardless of whether leaves are extracted with methanol-acetone-water (as in our work) or with ethanol/aqueous ethanol, the core phenolic constituents remain largely the same. Only minor qualitative differences have been noted in some cases (such as the presence of kaempferol-3-O-glucoside and delphinidin glycosides in the Egyptian samples) [6], which can be attributed to variations in plant origin, post-harvest handling, extraction techniques, and analytical methods. Overall, the convergent evidence from various studies confirms that P. cattleyanum leaves consistently produce a rich array of polyphenols—chiefly gallic/ellagic acid derivatives and flavonoid glycosides—that underpin their biological activities.

2.2. Biological Activity Relevant to Skin Health

Given the significant role of oxidative stress in the progression of many diseases, including various skin conditions [18], recent studies have focused on evaluating the antioxidant properties of plant extracts. In this study, we assessed the in vitro antioxidant, anti-collagenase, anti-elastase, anti-tyrosinase, antibacterial, and cytotoxic activities of P. cattleyanum leaf extract.

2.2.1. Antioxidant Activity

Antioxidant activity was examined using microplate-based assays in cell-free systems. Our findings suggest that P. cattleyanum leaf extract demonstrated a strong capacity to neutralize ABTS^•+^ and DPPH^•^ radicals, respectively. The antioxidant activity observed in the P. cattleyanum leaf extract was remarkably high, consistent with its polyphenol-rich composition. In the DPPH and ABTS assays, the extract exhibited strong free radical-scavenging ability, comparable to or exceeding that of standard antioxidants. For the DPPH^•^ assay, the Trolox equivalent (TE) content was 0.54 mg TE/g of dry extract. In the ABTS^•+^ assay, the TE value was 535.62 mg TE/g of dry extract (Table 3). This finding is consistent with those of previous studies on P. cattleyanum. The results of free radical scavenging activity for leaf extracts and the essential oil of P. cattleyanum collected in Brazil showed that the ethanolic extract exhibited the highest antioxidant activity, with scavenging percentages ranging from 94.00 to 94.57%. The aqueous extract exhibited scavenging activity between 91.58 and 92.62%. In contrast, the essential oil exhibited markedly lower antioxidant activity, with values ranging from 4.01 to 16.19% [19]. González-Silva et al. (2022) reported that P. cattleyanum leaf extracts obtained via optimized ultrasound-assisted extraction possess superior antioxidant capacity, significantly higher than that of conventionally extracted samples [8]. They identified numerous phenolic acids (gallic, protocatechuic, chlorogenic, p-coumaric, etc.) and the flavonol kaempferol in the leaves [8], all of which are known contributors to antioxidant efficacy. Similarly, Ribeiro et al. (2014) demonstrated that araçá fruit pulp and skin extracts efficiently scavenge a range of physiologically relevant reactive oxygen and nitrogen species [7].

The present study complements these reports by demonstrating that leaves, like fruits, are a potent source of natural antioxidants. The high radical-scavenging activity of our extract can be attributed to the synergistic effects of its constituent polyphenols. Ellagic acid and its glycosides, which were detected in this study, are powerful antioxidants capable of quenching free radicals and chelating redox-active metal ions [20]. Flavonoids, such as quercetin derivatives, are also well known for their strong antioxidant properties, functioning as hydrogen or electron donors to stabilize reactive species [21]. The abundance of these compounds in P. cattleyanum leaves explains its high total antioxidant capacity. This activity is particularly relevant to dermatological applications, as oxidative stress is a major driver of skin aging and inflammation, and P. cattleyanum extracts can neutralize reactive oxygen species, suggesting that they could protect skin cells from oxidative damage. The traditional use of the fruit to “fend off colds and infections” may partly stem from its antioxidant vitamin C and polyphenol content, which bolster the body’s defenses [1]. This study substantiates the reputation of P. cattleyanum as a health-promoting plant and highlights its potential as an antioxidant source for skin health.

2.2.2. Enzyme Inhibitory Activity

P. cattleyanum leaf extract was evaluated for its inhibitory activity against three enzymes involved in skin aging processes: collagenase, elastase, and tyrosinase. These enzymes play critical roles in the degradation of structural components and in melanogenesis, and their inhibition is considered a desirable feature for anti-aging and skin-brightening applications [22]. To our knowledge, this is the first report of P. cattleyanum collagenase, elastase, and tyrosinase inhibitory activities, particularly for specimens from Cabo Verde.

Collagenase Inhibition

Collagenase is a matrix metalloproteinase responsible for the degradation of collagen fibers, leading to reduced skin firmness and the formation of wrinkles [23]. At a concentration of 100 μg/mL, the extract showed strong collagenase inhibitory activity (90.37 ± 0.08%), which was slightly higher than that of the positive control, EGCG (89.59 ± 0.05%). This pronounced inhibitory effect suggests a strong potential of the extract to protect the extracellular matrix and maintain skin structural integrity. The observed activity may be attributed to the high content of flavonoids and phenolic compounds present in the extract. Flavons such as quercetin and kaempferol, detected mainly as glycosides, are known to inhibit collagenase by chelating the Zn^2+^ ion in the enzyme’s active site or by forming enzyme-substrate complexes, thereby suppressing enzymatic activity [24,25]. Additionally, ellagitannin and gallic acid derivatives may enhance this effect through their protein-binding properties, which can directly impede enzyme function.

Elastase Inhibition

Elastase in another proteolytic enzyme implicated in skin aging, as it degrades elastin fibers responsible for skin elasticity and resilience [26]. In contrast to its strong anti-collagenase activity, the P. cattleyanum leaf extract exhibited relatively weak inhibition of elastase. At 100 μg/mL of extract, elastase inhibition reached 58.13 ± 1.08%, whereas the positive control EGCG showed a significantly higher inhibitory effect (73.58 ± 0.12%). Although moderate, this level of elastase inhibition still indicates some potential to slow elastin degradation and may contribute synergistically to the overall anti-aging profile of the extract.

Tyrosinase Inhibition

Tyrosinase plays a central role in melanogenesis by catalyzing the hydroxylation of monophenols and the oxidation of o-diphenols to o-quinones, leading to melanin production and, consequently, to skin hyperpigmentation [22,27]. At a concentration of 100 μg/mL, the P. cattleyanum leaf extract inhibited mushroom tyrosinase activity by 61.09 ± 1.17%, indicating moderate inhibitory potency. In comparison, the reference inhibitor kojic acid tested at the same concentration achieved a markedly higher inhibition of 96.09 ± 0.29%. Despite being less potent than kojic acid, the tyrosinase inhibition observed for the extract is noteworthy, particularly given its natural origin. This activity can be attributed to phenolic constituents such as gallic acid, ellagic acid, and quercetin derivatives, which are known to inhibit tyrosinase through copper chelation at the enzyme’s active site or by acting as substrate analogues.

Overall, the P. cattleyanum leaf extract demonstrated a multi-target inhibitory profile relevant to skin health, characterized by strong collagenase inhibition, moderate elastase inhibition, and moderate tyrosinase inhibition. To our knowledge, this is the first report documenting the enzyme inhibitory activities of P. cattleyanum leaves. Although no previous studies have investigated this species in this context, the observed effects are consistent with reports on the closely related species Psidium guajava L., whose leaf extracts have shown potent inhibition of collagenase, elastase, and tyrosinase, along with general anti-aging activity [28]. The combined inhibition of enzymes involved in extracellular matrix degradation and melanogenesis, together with the strong antioxidant capacity of the extract, highlights the potential of P. cattleyanum leaves as a promising natural ingredient for anti-aging and skin-brightening formulations. A summary of the inhibitory activities of the P. cattleyanum leaf extract against collagenase, elastase, and tyrosinase, in comparison with the corresponding positive controls, is presented in Table 4.

2.3. Antibacterial Activity Against Skin Pathogens

The increasing resistance to conventional pharmaceutical drugs, along with their unexpected side effects, has prompted researchers to investigate plants as potential sources of novel antimicrobial agents. The antibacterial properties of plant extracts intended for topical application are particularly relevant, as many skin conditions, such as acne, can lead to wounds and secondary infection. Recent studies have emphasized not only the antimicrobial activity of these plant extracts but also their interactions with other cosmetic ingredients and their role within the complex network of the skin microbiome [29].

2.3.1. Agar Diffusion Assay

P. cattleyanum leaf extract was tested against bacteria commonly involved in acne and other skin-related infections. The larger the growth inhibition zone, the stronger the antibacterial properties of the extract, preferably >10 mm (Table 5). The sample exhibited good activity against microaerobic strains causing acne and seborrheic skin diseases, in the range of 21–12 mm. Quercetin used as positive control produced moderate inhibition zones against S. aureus and C. acnes but showed very weak activity against E. coli (Gram-negative), whereas sparfloxacin exhibited large inhibition zones against all three tested bacteria, consistent with its broad-spectrum antibacterial activity, confirming that the antibiotic reference standard represents the strongest antibacterial agent in the assay.

The antimicrobial activity of P. cattleyanum leaf extract observed in this study further enhances its potential for managing skin disorders. The extract exhibited broad-spectrum antibacterial effects, particularly against Gram-positive bacteria, which are relevant to skin health. We found that it inhibited the growth of Staphylococcus aureus and Cutibacterium acnes, which are common pathogens in skin infections and acne vulgaris. This finding is consistent with earlier reports that P. cattleyanum exhibits notable antimicrobial properties. Medina et al. (2011) demonstrated that araçá fruit extracts have antimicrobial activity against a range of bacteria and fungi, correlating with their high phenolic content [30]. More recently, El-Deeb et al. (2025) showed that a defatted methanolic leaf extract rich in polyphenols was effective against diarrhea-causing microbes: in their study, P. cattleyanum leaf phenolics produced clear inhibition zones (10–25 mm) against Escherichia coli, Salmonella enterica, Listeria monocytogenes, and S. aureus [4]. Our results are consistent with these findings, confirming that P. cattleyanum leaf constituents can suppress pathogenic bacteria. This is particularly significant in the context of acne. Acne lesions involve C. acnes (an anaerobic bacterium that contributes to inflammation) and often lead to secondary infection or colonization by S. aureus. By inhibiting these microorganisms, P. cattleyanum extract can reduce the bacterial load on the skin, thereby alleviating one of the root causes of acne and other infectious skin conditions. Additionally, the antimicrobial action of the extract complements its anti-inflammatory effects. Beltrame et al. (2022) reported that P. cattleyanum leaf extracts exhibit anti-inflammatory effects by inhibiting neutrophil chemotaxis, and they isolated hyperoside, miquelianin, and quercitrin as the flavonoid compounds responsible for this activity [9]. These flavonoids are present in our extract, and along with other constituents (e.g., verbascoside and galloylated quercetin glycosides), they likely contribute to modulating inflammation. Thus, P. cattleyanum addresses two key aspects of acne pathology: controlling bacteria and dampening inflammatory responses (such as the recruitment of immune cells and the release of pro-inflammatory mediators in the skin). This dual action, antimicrobial and anti-inflammatory, is highly beneficial in treating acne and similar dermatological disorders. This provides a scientific explanation for the traditional use of P. cattleyanum in infected wounds or inflammatory conditions, as the plant’s phytochemicals simultaneously combat infection and promote inflammation resolution, aiding the healing process.

2.3.2. Minimum Inhibitory Concentration (MIC) Results

The aim of determining MIC is to identify the lowest concentration of an antimicrobial agent that inhibits visible growth of a microorganism. The lower the MIC value, the greater the antibacterial activity of the tested extract.

The MIC determination results confirmed the findings of the agar diffusion assay. P. cattleyanum leaf extract exhibited strong antibacterial activity, with MIC values ranging from 125 to 1000 µg/mL. MIC values are reported in Table 6 together with the calculated MBC/MIC ratios, which allow distinction between bactericidal (ratio ≤ 4) and bacteriostatic (ratio > 4) effects. This analysis provides context for the potential clinical relevance of the tested antimicrobial agents. The extract showed a bacteriostatic effect and did not exhibit bactericidal activity. The gold standard for antibacterial activity remains a clinically relevant antibiotic; in this study, sparfloxacin was used as the reference control. Its MIC against acne-associated strains ranged from 0.26 to 4.15 µg/mL, confirming its strong and consistent activity in vitro. In comparison, the MIC of reference quercetin ranged from 500 to more than used in assay 1000 µg/mL, demonstrating moderate antibacterial activity as a natural reference compound.

2.3.3. Interactions Between P. cattleyanum Extract and Antibiotics Against Ane-Associated

The leaf extract of P. cattleyanum, when used in conjunction with antibiotics commonly used to treat skin infections (Sparfloxacin, Cefepime, Ceftriaxone), did not exhibit antagonistic effects. Importantly, it demonstrated either complete or partial synergism or no interaction (Table 7). The observed beneficial synergistic effects may allow a reduction in the antibiotic dose required to achieve an antibacterial effect and, consequently, may decrease the risk of antibiotic resistance, benefiting both patient health and healthcare systems. The World Health Organization warns that antibiotic resistance represents a global threat and that if current antibiotic-use practices are not changed, by 2050, antimicrobial resistance could potentially result in one death every three seconds from infections that are currently effectively treatable, making it a more frequent cause of death than cancer [31].

2.4. Cytotoxic Properties

The cytotoxicity of the P. cattleyanum leaf extract (PC) was assessed in Vero cells after 24 h of incubation using the MTT assay (Figure 2). The PC extract did not significantly reduce cell viability at concentrations up to 250 µg/mL, with viability remaining approximately 100% compared to the control. A statistically significant reduction in cell viability (p < 0.0001) was observed at higher concentrations, with approximately 66% and 49% viability at 500 µg/mL and 1000 µg/mL, respectively.

Four-parameter nonlinear regression analyses of the log_10_-transformed concentrations allowed estimation of the IC_50_, which the software indicated to be approximately 2.673 (~470.99 μg/mL); however, this value should be considered an approximate estimate rather than an exact parameter. IC_90_ could not be reliably determined because the maximal reduction in cell viability did not reach 10%, and calculation would require extrapolation beyond the experimental data.

Overall, these results suggest that PC extract exhibits broad in vitro efficacy, with only moderate cytotoxic effects observed at the highest tested concentrations, indicating limited cytotoxicity within the experimental range.

Comparing our results with those obtained by other researchers, Houël et al. (2015) evaluated the cytotoxic effects of an aqueous extract of Psidium acutangulum Mart. ex DC. and reported moderate activity, with IC_50_ values exceeding 100 μg/mL [32]. In contrast, Houël et al. (2016) assessed an ethyl acetate fraction of P. acutangulum and observed IC_50_ values above 57.4 μg/mL [33]. Furthermore, Machado et al. (2018) demonstrated that a leaf decoction of P. brownianum Mart. ex DC. collected in Brazil exhibited higher toxicity, resulting in 90.85% fibroblast cell death, followed by a hydroethanolic leaf extract of P. guajava, which caused 76.30% cell death [34].

Considering the importance of cytotoxicity assessment in new drug development, there is low cytotoxicity data for P. cattleyanum [35]. Generating such data would strengthen the existing body of knowledge and help position this species as a potential plant-derived medicinal drug for treating various diseases.

2.5. Evaluation of Physicochemical and Organoleptic Properties

The tested cream with PC (P. cattleyanum leaf extract) met the expected physicochemical and organoleptic quality criteria. The applied assessment procedures were performed according to previously published methodologies [36,37], confirming the proper consistency, homogeneity, and overall stability of the extract-containing formulation.

The centrifugation assay revealed no signs of phase separation at any of the evaluated storage time points, confirming the physical stability of the tested cream. Sensory assessment indicated a neutral odor and beige color characteristic of the incorporated extract. The formulation exhibited a smooth and homogeneous texture without detectable particulate matter or crystallization of fatty alcohols. The spreadability was rated as good, with uniform distribution on the skin, rapid absorption, and no greasy or sticky residues. No adverse sensory attributes, such as unpleasant chemical odor or coarse texture, were observed during the evaluation.

Overall, the physicochemical and sensory analyses demonstrated that the prepared cream maintained satisfactory stability and homogeneity and was well accepted with respect to appearance, odor, and application properties.

The mean droplet diameter of the dispersed oil phase is a critical parameter that influences the physical stability and functional performance of cosmetic emulsions [31]. A finer and more homogeneous droplet distribution enhances kinetic stability by minimizing separation phenomena, such as creaming and coalescence, which positively affect the appearance and sensory attributes of the emulsions [37,38].

Microscopic evaluation confirmed that all formulations exhibited a uniform distribution of droplets without visible signs of aggregation or phase separation over the 90-day storage period. The mean droplet diameters ranged from approximately 4.82 to 5.23 µm, which is typical of stable oil-in-water (O/W) emulsions prepared under moderate homogenization conditions [37].

For samples stored under refrigerated conditions (4 °C), the Kruskal-Wallis test revealed statistically significant differences in droplet diameter distributions across storage time points (H(5, N = 900) = 30.14, p < 0.001). The results of the non-parametric analysis for the droplet size distribution are presented in Table 8. Analysis of the mean ranks indicated that the highest droplet diameters were observed on day 30 (mean rank = 523.7), whereas the lowest values were recorded on day 90 (mean rank = 368.2). Importantly, the droplet diameters measured after 90 days were comparable to those observed at the initial time point, and no monotonic increase in droplet size over time was detected. The median test confirmed statistically significant differences between the time points (χ^2^ = 29.81, df = 5, p < 0.001). A transient shift toward values above the overall median (4.88) was observed on day 30, whereas the measurements on day 90 were predominantly below the median. These results indicate the presence of temporary fluctuations in droplet size, without evidence of progressive coalescence.

In contrast, no statistically significant differences in droplet diameter distribution were observed for cream with PC (P. cattleyanum leaf extract) stored at 20 °C. The Kruskal-Wallis test showed no significant effect of storage time (H(5, N = 900) = 3.33, p = 0.650). Mean ranks remained comparable across all analyzed time points, indicating a stable droplet size distribution throughout the 90-day storage period. Similarly, the median test did not reveal statistically significant differences between time points (χ^2^ = 5.07, df = 5, p = 0.408), confirming the absence of systematic shifts in the droplet diameter relative to the overall median value (4.81).

The mean Sf values of the tested creams ranged from approximately 15.73 × 10^3^ to 16.38 × 10^3^ g·cm^2^, indicating good spreading performance and satisfactory application properties [37,39,40].

The Shapiro–Wilk test confirmed the normal distribution of the spreadability (Sf) data (p > 0.05). Homogeneity of variances was verified using Levene’s test (p > 0.05). Therefore, a one-way ANOVA was performed. No statistically significant differences in spreadability were observed across the analyzed conditions (F(10,22) = 0.84, p = 0.60), indicating the stable application properties of the formulation during the study period.

Over the 90-day rheological study of cream with PC, the apparent viscosity at a Dr of 1 s^−1^, 5 s^−1^, and 100 s^−1^, the consistency index (K), the flow behavior index (n), and thixotropy (A) remained stable regardless of storage temperature (4 °C vs. 20 °C) and time (1, 7, 14, 30, 60, 90 days). Statistical analysis of the consistency index (K), derived from the Ostwald-de Waele model, revealed no significant effects of storage time or temperature over the 90-day study period. One-way ANOVA showed no statistically significant differences between the groups (F(10,22) = 0.46, p = 0.897), confirming the stability of the internal structure of the system.

The pH of the formulation remained stable throughout the study, with mean values ranging from 6.33 to 6.43 (Table 9). One-way ANOVA did not reveal statistically significant differences in pH between the analyzed conditions (F(10,22) = 1.67, p = 0.152), confirming the stability of pH during storage. No significant pH fluctuations were observed at any time point, indicating that the formulation maintained chemical stability and that no degradation processes affecting acidity occurred during storage.

The absence of phase separation in creams with PC samples across all storage intervals indicates that the formulations maintained their structural integrity throughout the testing period. This consistent resistance to centrifugal stress confirms that the emulsions exhibit high physical stability and are unlikely to undergo destabilization processes, such as creaming or coalescence, during storage [37,41].

Spreadability reflects the ease of application of semisolid formulations and serves as an indirect indicator of rheological characteristics. Higher Sf values are associated with a softer, more plastic texture and appropriate viscosity, enabling a uniform distribution on the skin [39].

In the present study, the mean Sf values obtained for both emulsions ranged from 71–78 × 10^3^ g·cm^2^, indicating good spreadability and favorable application properties of the tested formulations. These findings were consistent with the results of the authors’ own sensory evaluation, which confirmed easy application and satisfactory user acceptability [37]. The lack of statistically significant changes over the storage period indicated the preservation of rheological properties and sustained structural integrity of the emulsions.

The unchanged Sf values throughout storage reflect the preserved emulsion structure, attributable to a balanced oil–water phase ratio and appropriate emulsifier choice, in agreement with previous reports on stable O/W systems [37,39].

The cream with PC demonstrated shear-thinning behavior accompanied by stable and moderate thixotropy, as evidenced by a consistent hysteresis loop area (A), which is characteristic of well-designed topical semisolid formulations [37,42,43]. The cream with PC showed stable pseudoplastic and moderately thixotropic behavior, with high viscosity at low shear (1 s^−1^) and a pronounced decrease at higher shear rates (5 and 100 s^−1^), facilitating spreading during application and structural recovery afterward. The unchanged viscosity profile, consistency index (K), and hysteresis loop area (A) throughout the storage period and at both temperatures indicated a preserved interfacial network and a well-balanced emulsifier–thickener system, without structural degradation or excessive densification [39,42,43].

Stable pH values throughout the 90-day storage period indicated that the extracts did not induce acidification or alkalinization of the formulations. The measured pH range (6.33–6.43) is acceptable for topical use and is close to the physiological skin pH, supporting chemical stability and skin compatibility [44,45]. The preservation of this mildly acidic milieu, known as the acid mantle, is crucial for barrier integrity, microbiome balance, and enzyme-mediated lipid processing in the skin [44].

Therefore, the pH of the cream with PC (P. cattleyanum leaf extract) is compatible with skin physiology, and its stability during storage further confirms its chemical integrity and suitability for topical application.

3. Materials and Methods

3.1. Chemicals and Reagents

Acetonitrile, formic acid, and water were supplied for LC analysis, as well as thiazolyl blue tetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), and hydrochloric acid by Merck (Darmstadt, Germany). All other chemicals were of analytical grade and obtained from the Polish Chemical Reagent Company (POCH, Gliwice, Poland). 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^•^), 2,2′-azino-bis-(3-ethyl-benzothiazole-6-sulfonic acid) (ABTS^•+^), collagenase from Clostridium histolyticum, elastase from porcine pancreas, (-)-epigallocatechin gallate (EGCG), Folin–Ciocalteu reagent, N-[3-(2-Furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA), N-Succinyl-Ala-Ala-Ala-p-nitroanilide (SANA), ethylenediaminetetraacetic acid disodium salt (Na_2_EDTA), and Trolox (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), penicillin-streptomycin solution, thiazolyl blue tetrazolium bromide, hydrochloric acid, and dimethyl sulfoxide (DMSO) were obtained from Merck (Darmstadt, Germany). Cutibacterium acnes PCM 2334, C. acnes PCM 2400, Escherichia coli (w.c.i.), and Pseudomonas aeruginosa (w.c.i.) were purchased from the Hirszfeld Institute of Immunology and Experimental Therapy, PAN, Lublin, Poland, while Staphylococcus epidermidis ATCC 12228 and S. aureus ATCC 25923 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Müeller-Hinton agar (MH-agar) or broth (MH-broth) were obtained from Oxoid Ltd. (Basingstoke, Hampshire, UK), while Brain Heart Infusion agar or broth (BHI-agar, BHI-broth) was obtained from Bio-Maxima S.A. (Lublin, Poland). GSC (Glyceryl Stearate Citrate), Stearic Acid (Stearic Acid), Caprylic/Capric Triglyceride (Caprylic/Capric Triglyceride), Isopropyl Myristate (Isopropyl Myristate), Isopropyl Palmitate (Isopropyl Palmitate), Cetiol^®^ Ultimate (Undecane (and) Tridecane), Dicaprylyl Ether (Dicaprylyl Ether), Cetiol^®^ Sensoft (Propanediol Dicaprylate), Sodium Polyacrylate (Sodium Polyacrylate), Propanediol (Propanediol), Pentylene Glycol (Pentylene Glycol), Glycerin (Glycerin), Sodium Phytate (Sodium Phytate), Panthenol (Panthenol), Vitamin E (Tocopherol) were obtained from BASF. Glyceryl Monostearate (Glyceryl Stearate), Shea Butter (Butyrospermum Parkii Butter) were obtained from Croda. TEGO Care CG 90 (Cetearyl Glucoside (and) Cetearyl Alcohol), Behenyl Alcohol (Behenyl Alcohol), Cetearyl Alcohol (Cetearyl Alcohol), Dimethicone 5 cSt (Dimethicone), TEGO^®^ Care OP (Polyglyceryl-3 Stearate), Plant Extract (Plant Extract), Phenoxyethanol (and) Ethylhexylglycerin (Preservative Blend)—Evonik, Cera Alba (Beeswax), Xanthan Gum (Xanthan Gum), Aqua (Purified Water) were obtained from Carl Roth. The Vero cell line (CCL-81) and EMEM medium were purchased from ATCC (Manassas, VA, USA), and fetal bovine serum (FBS) was purchased from PAN-Biotech (Aidenbach, Germany).

3.2. Plant Material

Leaves of P. cattleyanum Sabine were collected in Santo António das Pombas, Santo Antão, Cape Verde (coordinates 17°08′29″ N; 25°02′21″ W), in July 2023. Taxonomical identification was confirmed by Prof. Arlindo Rodrigues Fortes. A voucher specimen was deposited at the Escola Superior de Ciências Agrárias e Ambientais (PC-0723).

3.3. Preparation of the Extracts

The collected plant material was air-dried at an average temperature of 24.0 ± 0.5 °C in the shade to a constant weight and then powdered. Dried P. cattleyanum leaves (35.00 g) were used for extraction. The powdered material was extracted thrice, each time with 350 mL of a methanol, acetone, and water (3:1:1; v/v/v) mixture. Each extraction was performed by sonication in an ultrasonic bath at a controlled temperature of 45 ± 2 °C for 30 min. The combined filtrates were concentrated under reduced pressure (rotary evaporation at ~40 °C until dryness), then frozen, and lyophilized using a vacuum concentrator (Free Zone 1 apparatus; Labconco, Kansas City, KS, USA) to yield a dried crude extract (32.11 g, representing ~91.7% of the initial dry material; drug-to-extract ratio ~1:0.92 w/w). All solvents were effectively removed during evaporation and freeze-drying, leaving no detectable residual methanol or acetone in the final extract.

3.4. Total Flavonoid, Phenolic and Phenolic Acids Content

The total phenolic content (TPC) and total flavonoid content (TFC) were quantified using colorimetric assays, as described by Szewczyk et al. (2020) [12]. Absorbance was measured at 680 and 430 nm, respectively, using a Pro 200F ELISA Reader (Tecan Group Ltd., Männedorf, Switzerland).

The TPC was calculated from a gallic acid calibration curve (R^2^ = 0.9992) prepared in the concentration range of 0.002–0.16 mg/mL, and the results were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry extract (DE). TFC was determined from a quercetin calibration curve (R^2^ = 0.997) within the concentration range of 0.004–0.2 mg/mL, and the results were expressed as milligrams of quercetin equivalents (QE) per gram of DE.

The total phenolic acid content (TPAC) was assessed using Arnov’s reagent according to the Polish Pharmacopoeia XIII (official translation of Ph. Eur. 11.0) [46]. Absorbance was recorded at 490 nm, and TPAC was calculated from a caffeic acid calibration curve (R^2^ = 0.9745) in the range of 3.36–23.52 µg/mL. The results are expressed as milligrams of caffeic acid equivalents (CAE) per gram of DE.

3.5. HPLC-DAD-MS Analysis

HPLC-DAD-MS/MS analyses were conducted using an Ultimate 3000 series system equipped with DAD detection and coupled to an Amazon SL ion trap mass spectrometer (Bruker Daltonik GmbH, Bremen, Germany). Separation was performed on a Kinetex XB-C18 column (150 mm × 2.1 mm × 1.7 µm; Phenomenex Torrance, CA, USA). The column was maintained at 25 °C. Elution was performed using a mobile phase consisting of A: water (0.1% formic acid, v/v) and B: acetonitrile (0.1% formic acid, v/v), following a three-step gradient (%B): 0 min at 1%, 60 min at 26%, and 70 min at 50%. The flow rate was set at 0.3 mL/min. The injection volume was 2 µL. Between the injections, the column was equilibrated for 7 min. The UV-Vis spectrum was recorded within the 200–450 nm range, with chromatograms acquired at 254 nm. Mass spectra were recorded in the negative ion mode. The proposed identification of the detected compounds was done based on a literature search in the Reaxys database. Whenever possible, comparisons with chemical standards were made. All standards were previously isolated in the Department of Pharmaceutical Biology, and their identities were confirmed by NMR analysis.

3.6. Antioxidant Activity

3.6.1. DPPH• Radical Scavenging Assay

The 2,2-diphenyl-1-picrylhydrazyl (DPPH^•^) free radical scavenging activity of P. cattleyanum leaf extract was determined according to the method described by Olech et al. (2020), with some modifications [47]. The decrease in DPPH^•^ absorbance induced by the extracts was monitored at 517 nm after 30 min of incubation at 28 °C. The results were calculated from individual sample measurements and expressed as milligrams of Trolox equivalents per gram of dry extract (mg TE/g DE).

3.6.2. ABTS•+ Radical Cation Decolorization Assay

The antioxidant capacity of P. cattleyanum leaf extract was assessed using the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS^•+^) decolorization assay, as described by Pieczykolan et al. (2022) [48]. Absorbance was measured at 734 nm after a 6 min incubation, and the results were expressed as milligrams of Trolox equivalents per gram of dry extract (mg TE/g of DE).

3.7. Enzyme Inhibitory Activity

3.7.1. Anti-Elastase Activity

Anti-elastase activity was determined spectrophotometrically according to Chiocchio et al. (2018) [27], with minor modifications. Porcine pancreatic elastase (3.33 mg/mL) and P. cattleyanum leaf extract (5–100 μg/mL) were incubated in Tris-HCl buffer (1.6 mM, pH 8.0) for 15 min at 29 °C. The reaction was initiated by adding N-succinyl-Ala-Ala-Ala-p-nitroanilide (AAAPVN, 2 mM) as the substrate. The final reaction mixture (200 μL) in a 96-well microplate contained buffer, 0.8 mM AAAPVN, 1 μg/mL elastase, and 25 μL of the extract at the desired concentration. After 20 min of incubation, absorbance was measured at 410 nm. Epigallocatechin gallate (100 μg/mL) was used as a positive control.

3.7.2. Anti-Collagenase Activity

Anti-collagenase activity was evaluated using N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala (FALGPA) as the substrate, as described by Mandrone et al. (2018) [49]. Collagenase from Clostridium histolyticum (20 mU), prepared in Tricine buffer (50 mM, pH 7.5) at an initial concentration of 0.8 U/mL, was incubated with varying concentrations of P. cattleyanum leaf extract for 15 min at 35 °C. The final reaction mixture (150 μL) contained Tricine buffer, 0.8 mM FALGPA, 0.1 U collagenase, and the appropriate concentration of extract. The absorbance was recorded at 345 nm. Epigallocatechin gallate (100 μg/mL) was used as the positive control for the assay.

3.7.3. Anti-Tyrosinase Activity

Anti-tyrosinase activity was determined according to the method described by Zengin et al. (2014), with slight modifications [50]. Various concentrations of P. cattleyanum leaf extract (20 μL) were mixed with mushroom tyrosinase solution (200 U/mL, 40 μL) and phosphate buffer (100 μL, 50 mM, pH 6.5) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was initiated by adding L-DOPA (L-3,4-dihydroxyphenylalanine; 40 μL, 0.5 mM) and incubated for 15 min. Absorbance was measured at 475 nm relative to that of a control without the inhibitor. In the control, phosphate buffer replaced the extract and represented 100% of the enzyme activity. Tyrosinase inhibition was calculated by comparing the enzyme activity in the presence and absence of the extract. Kojic acid (100 μg/mL) served as the standard inhibitor.

3.8. Antibacterial Activity Assays

3.8.1. Bacterial Strains and Their Maintenance

Microaerobic Gram-positive Cutibacterium acnes PCM 2334 and C. acnes PCM 2400 (as representatives of acne strains), and aerobic Gram-positive Staphylococcus epidermidis ATCC 12228, S. aureus ATCC 25923, and aerobic Gram-negative Escherichia coli (w.c.i.) as well as Pseudomonas aeruginosa (w.c.i.) clinical isolates from infected wounds (w.c.i.) (possessed from the Hirszfeld Institute of Immunology and Experimental Therapy, PAN, Poland) were used in this study. All strains were properly stored at the Chair and Department of Biochemistry and Biotechnology of the Medical University of Lublin, Poland. Müeller-Hinton agar or broth [MH-agar or MH-broth) and BHI-agar or BHI-broth (Oxoid Ltd. (UK)] were used for aerobic strains, and brain Heart Infusion agar or broth [bHI-agar, bHI-broth; bio-Maxima S.A. (Poland)] for microaerobic strains culture, respectively. Inoculum was prepared by subculturing bacteria in solid medium (MH or BHI) at 37 °C for 24 h or 48 h, respectively.

The antibacterial activity of the plant extracts was evaluated against positive controls, including Quercetin dihydrate (Sigma-Aldrich, St. Louis, MO, USA; catalog number Y0001009, purity ≥ 98%) as a natural antibacterial reference, and Sparfloxacin (Pol-Aura, Poland) as a clinically relevant topical antibiotic for skin pathogens.

The microbiological activity assays were performed in triplicate, and the results are presented as mean ± standard deviation (SD).

3.8.2. Disc Diffusion Method

The antibacterial activity of P. cattleyanum leaf extract was determined using a modified agar disc diffusion procedure [51]. To perform an initial screening test, extract solutions with a concentration of 10 mg/mL in DMSO were prepared. The next stage was the preparation of Petry plates. They were prepared by pouring MH-agar (aerobic bacteria) or BHI agar (microaerobic bacteria) according to the manufacturer’s instructions.

A bacterial inoculum with a colony concentration of 1.5 × 10^8^, that is 0.5 McFarland, was prepared and bacterial suspensions were inoculated on prepared Petry plates using a sterile cotton swab. The solutions of the tested extract were applied to each marked place in an amount of 10 µL, which, taking into account the concentration of the stock solution, constituted 100 µg. The plates were incubated to provide optimal growth conditions for the microorganisms as follows: aerobic bacteria—incubation at 37 °C for 24 h, under aerobic conditions; microaerobic bacteria—incubated at 37 °C for 48 h, ensuring anaerobic conditions. After the incubation period, the zones of bacterial growth inhibition around the studied extract were measured using a microbiological ruler.

3.8.3. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) Determination

MIC is the lowest concentration of the tested extract that inhibits the growth of microorganisms. The MIC test was performed using the double dilution method in 96-well plates. A total of 380 µL of MH broth (for aerobic bacteria) or BHI broth (for microaerobic bacteria) was placed in the first row of the plate. Then, 200 µL of the broth was poured into the remaining wells of the plate. Then, 18 µL of the solution of the tested extract was added to the appropriate columns of the first row, obtaining a range from 1600 µg/mL to 6.25 µg/mL. Next, 200 µL of the resulting medium was transferred, starting from the first row, with double dilutions. Then, 2 µL of the previously prepared bacterial inoculum with a concentration of 0.5 McFarland was added to each well. The experiment included columns for positive control (assessing the growth of bacteria), negative control (assessing the sterility of the experiment), and reagent control (considering the color of the extract in subsequent dilutions). MBC is defined as the lowest concentration of a compound that exhibits bactericidal activity. To determine the MBC value, a 96-well plate was used, in which the MIC of the compounds was determined in the previous step. The 10 μL of medium was taken from clear wells in which no turbidity, that is, no bacterial growth, was observed and transferred with a pipette to a clean Petry’s plate with the selected agar. The plates were then incubated under appropriate conditions to assess the sterility of the culture or appearance of bacterial colony growth. The value of the bactericidal concentration (MBC) was considered to be the concentration at which no bacterial growth was visible in the medium. Therefore, it is advisable to determine the value of the MBC/MIC ratio to determine the bactericidal or bacteriostatic nature. Bactericidal compounds kill microbial cells, whereas bacteriostatic compounds only change the metabolism of bacterial cells, preventing their growth and multiplication. According to the Clinical and Laboratory Standards Institute, a compound is considered to have bactericidal activity when the MBC/MIC ratio is ≤4, and when the MBC/MIC ratio is ≥8, the drug is indicated as bacteriostatic.

3.8.4. Determination of Interactions Between P. cattleyanum Extract and Antibiotics Against Acne-Associated Strains

The interactions between the plant extract and the following antibiotics were evaluated: ceftriaxone (Polpharma SA, Starogard Gdański, Poland), cefepime (Bristol-Myers Squibb, Sermoneta, Italy), and sparfloxacin (Dainippon Pharmaceutical Co., Ltd., Cheshire, UK). A checkerboard assay based on MIC determination was performed. Stock solutions and serial twofold dilutions of the antibiotics and extract were prepared up to four times the MIC value. A total of 50 µL of Mueller–Hinton broth was added to each well of microdilution plates. The first antibiotic in each combination was serially diluted vertically, whereas the P. cattleyanum leaf extract was diluted horizontally in a 96-well plate. Subsequently, 100 µL of bacterial inoculum at a concentration of 1.5 × 10^8^ CFU/mL was added, and the plates were incubated under the appropriate conditions.

The resulting checkerboard contained all possible combinations of the two agents tested. The plate layout also included MIC determination for each agent tested individually. For each concentration combination, the fractional inhibitory concentration index (FICI) was calculated using the following equation:

$$[eqn]$$

where MIC A is the MIC of agent A alone, MIC A/B is the MIC of agent A in combination with agent B, and MIC B and MIC B/A are defined similarly for agent B.

3.9. Evaluation of Cytotoxicity

Cytotoxicity testing was conducted using a normal mammalian cell line, Vero (African green monkey kidney cells from ATCC, cat no. CCL-81). The Vero cell line is widely used as an in vitro model to evaluate the cytotoxicity of medicinal and cosmetic compounds because of its reproducible growth characteristics and sensitivity to a range of chemical and biological agents, making it an important tool for preliminary safety assessment before advancing to in vivo studies [52,53,54,55]. Cells were cultured according to the manufacturer’s recommendations, i.e., in EMEM culture fluid supplemented with 10% FBS. Before the experiment, cells were seeded into 96-well plates at a concentration of 2 × 10^5^ cells/mL and incubated for 24 h at 37 °C. After this time, PC extract dilutions were prepared at concentrations of 1.95–1000 μg/mL and added to the cells. The cells were incubated for 24 h. Their metabolic activity was then assessed using the MTT assay. Cells treated with culture medium without PC extract were used as controls. Results are presented as mean viability values ± standard deviation per % of control (control was 100%). To assess statistical significance, an unpaired Student’s t-test was performed using GraphPad Prism, Version 5.04. To determine IC_50_ and IC_90_ values (inhibitory concentration that reduce cell viability to 50% and 90% compared to control, respectively), 4-parameter nonlinear regression analyses were performed (GraphPad Prism, Version 5.04).

3.10. Composition and Preparation of Cosmetic Cream

A cream formulation containing P. cattleyanum leaf extract (PC) dissolved in water (1:10; 100 mg/1 g) was developed according to our recipe. The cream was prepared in batches of 500 g. The composition of the cream is shown in Table 10.

3.11. Cream Preparation

The cream was manufactured using an IKA LR 1000 Control laboratory reactor equipped with a thermostatic heating system, mechanical stirrer, vacuum unit, and a rotor–stator homogenizer. Initially, the oil phase was weighed, transferred to the reactor vessel, hermetically closed, and heated to 70 °C under continuous slow agitation (50 rpm). Separately, the aqueous phase was heated to 50 °C on a hot plate and then gradually added to the oil phase. The combined system was subsequently heated until the total batch temperature reached 70 °C. At this point, the stirring speed was increased to 150 rpm, and a vacuum was applied to the chamber while simultaneous high shear homogenization was performed at 15,000 rpm. The conditions were maintained for 15 min. Afterwards, heating and high-shear mixing were stopped, and the batch was gently stirred at 50 rpm until it cooled to 40 °C. Upon reaching this temperature, the Phase C components were incorporated, after which the mixture was again homogenized and mixed under vacuum for an additional 15 min. After preparation, the creams were stored at room temperature until the following day, when they were subjected to initial analyses. Each formulation was divided into two 250 g portions and stored at 4 °C and 20 °C for further stability studies. The concentration of the P. cattleyanum leaf extract (PC) in the cream was (2 mg/1 g).

3.12. Evaluation of Physicochemical and Organoleptic Properties

The physicochemical properties of the samples were assessed according to the procedures described in the literature [36,37,38,39,40,41,44,45,56,57,58].

3.13. Centrifugation Test for Physical Stability

Approximately 1 g of each cream sample was transferred into Eppendorf-type centrifuge tubes and centrifuged using an Eppendorf 5418 R laboratory centrifuge. The centrifugation process was performed at a rotational speed of 9000 rpm, generating a relative centrifugal force (RCF) of approximately 7000× g for 10 min. Following centrifugation, the samples were visually inspected for any signs of phase separation or physical instability [36,37,41]. The analyses were performed at predetermined time points after 1, 7, 14, 30, 60, and 90 days of storage at two temperatures (4 °C and 20 °C). These time intervals were selected to enable comprehensive monitoring of the short- and intermediate-term stability of the emulsions [59]. Early assessments (days 1, 7, and 14) allowed the detection of rapid destabilization processes typical of freshly prepared emulsions, including coalescence, creaming, or phase separation, whereas later measurements (days 30, 60, and 90) facilitated the evaluation of long-term physical stability under standard storage conditions.

For each formulation and storage condition, three independent samples were analyzed in parallel at each time point to ensure the reproducibility of the results. The applied study design is consistent with the general stability testing principles described in the ICH Q1A(R2) guideline, which recommends a systematic evaluation of dosage forms at multiple time points to characterize stability trends and predict product shelf life [59].

3.14. Organoleptic Evaluation of the Preparation

Sensory evaluation was performed by the authors using a self-assessment protocol. The creams were examined with respect to odor, color, texture, and ease of application on the dorsal surface of the hand. Odor perception was categorized as pleasant, neutral/odorless, or unpleasant (chemical odor). The texture was classified as homogeneous (smooth consistency without detectable solid particles) or non-homogeneous when particulate matter, such as fatty alcohol crystals, could be perceived during application. Spreadability was evaluated by applying a small amount of the formulation to the volar area of the wrist and rubbing it onto the skin. Products with good spreadability were characterized by uniform distribution, smooth absorption, and the absence of streaking, clumping, pilling, or formation of greasy or sticky films. In contrast, poor spreadability was assigned to formulations that left visible streaks or lumps and/or a pronounced heavy residual layer on the skin surface [36,37].

3.15. Particle Size Evaluation

To monitor variations in droplet size within the dispersed phase, the emulsions were analyzed using bright-field optical microscopy (Leica ICC50 HD, Wetzlar, Germany) with a 40× air objective lens. For sample preparation, a thin, evenly distributed layer of cream was placed on a microscope slide and covered with a coverslip. At each time point, three randomly selected microscopic fields were examined for each formulation. A total of 150 oil droplets (3 × 50 droplets) were randomly measured per sample using the LAS EZ image analysis software v.4.13 (Leica Microsystems, Germany) [37,38]. Representative microscopic images were recorded with automated scale calibration using a 50 μm scale bar. For each formulation, the mean diameter of dispersed oil droplets was determined and subsequently compared across storage periods of 1, 7, 14, 30, 60, and 90 days to evaluate, via statistical analysis, potential droplet coalescence and progressive emulsion destabilization. Exemplary micrographs of the droplet morphologies (scale bar = 50 μm) are presented in Figure 3, while the corresponding droplet size distribution histograms are shown in Figure 4.

Statistical analyses were performed using MS Office 2021 (Excel) and Statistica 13.1 software. Variations in the diameter of dispersed oil droplets during storage were evaluated to assess emulsion physical stability. Prior to statistical testing, the data distribution was examined using the Shapiro-Wilk normality test. For all analyzed datasets, the p-values were below 0.05, indicating a non-normal distribution of droplet size data.

Given the lack of normality and the independent nature of the measurements collected at successive time points, non-parametric statistical methods were applied. Differences in droplet diameter across storage times were assessed using the Kruskal-Wallis rank-based analysis of variance, with storage time (days 1, 7, 14, 30, 60, and 90) treated as the grouping factor. Analyses were performed separately for samples stored at 4 °C and 20 °C temperature.

Additionally, the median test was used as a complementary approach to evaluate the differences in the distribution of droplet diameters relative to the overall median value. Statistical significance was set at p < 0.05.

3.16. Spreadability Test and Statistical Analysis of Spreadability Factor (Sf)

Spreadability was evaluated using an extensometer by recording the lateral expansion of the cream samples under standardized loading conditions [37,39,40]. All measurements were conducted at an ambient temperature (20–22 °C). Each cream sample (1.00 g) was placed centrally on a circular test plate and compressed using a flat-ended piston. Stepwise loading was applied using weights of 200, 400, 600, 800, and 1000 g, each maintained for 1 min. Following each loading step, the radius of the resulting film was recorded in four directions at angular intervals of 45 °C, and the values were averaged.

Each test sequence was repeated thrice for each formulation. Based on the calculated mean diameters, the corresponding spreading areas were determined for each load applied. The spreadability factor (Sf) was calculated as the area under the curve generated from a plot of the spreading area versus the applied load [37]. The obtained spreadability factor (Sf) values, expressed in g·cm^2^, allowed for a quantitative comparison of the ease of application among the tested formulations. All measurements were performed in triplicates for each cream. The results for the creams with PC are presented as mean ± standard deviation (SD) in Table 9.

The normality of the data distribution was assessed using the Shapiro-Wilk test, and the homogeneity of variances was verified using Levene’s test. As the assumptions for parametric testing were met, a one-way analysis of variance (ANOVA) was performed. The storage time and temperature were considered jointly as a single grouping factor. Statistical results are reported as F statistics with corresponding degrees of freedom [F(df_1_, df_2_)] and p-values, with statistical significance set at α = 0.05.

3.17. Rheological Testing and Statistical Analysis of Rheological Parameters K

The rheological characterization of the cream consistency and flow behavior was conducted using a rotational rheometer (Anton Paar RheolabQC) equipped with a concentric cylinder measuring system (CC27/S) and Peltier temperature control set to 25 °C. Data acquisition and analysis were performed using RheoCompass™ software version 1.30.1164 (Anton Paar, Austria). Prior to measurement, the samples were carefully introduced into the measuring cup and equilibrated for 10 min. A controlled shear rate program was applied, consisting of an ascending ramp from 0.1 to 100 s^−1^, followed by a descending ramp from 100 back to 0.1 s^−1^. The hysteresis loop area between the upward and downward flow curves was calculated as an indicator of the thixotropic behavior. The apparent viscosity (η) values were extracted from the flow curves at defined shear rates of Dr (1, 5, and 100 s^−1^). The experimental data were fitted to the Ostwald–de Waele power-law model to determine the consistency coefficient K (Pa·s^n^) and flow behavior index n. Temporal variations in these parameters were used to assess potential aging-related changes in the rheological properties of the tested formulations. Each measurement was performed in triplicates for all samples. Representative flow curves are shown in Figure 5. All results are expressed as mean ± SD, with rheological evaluation based on the parameters K (consistency index), n (flow behavior index), and hysteresis loop area (A), representing thixotropy, as summarized in Table 9.

Statistical analysis was performed for the consistency index (K), which is considered the most sensitive parameter for reflecting structural changes in non-Newtonian systems. The normality of the K values was verified using the Shapiro-Wilk test, and homogeneity of variances was confirmed using Levene’s test. Consequently, a one-way analysis of variance (ANOVA) was applied to evaluate the effect of storage time and temperature on K. No statistically significant differences were observed (p > 0.05), indicating the stability of the consistency index during storage.

3.18. pH Measurement and Statistical Analysis of pH Measurements

The pH of the formulations was measured using a calibrated pH meter (Elmetron, Poland). For the analysis, 5 g of each cream was accurately weighed and dispersed in 50 g of distilled water to obtain a homogeneous suspension. The pH electrode was immersed directly into the prepared sample, and readings were taken after the signal was stabilized. All measurements were performed at 20–22 °C in triplicate for each formulation and storage condition at all scheduled time points. The results are presented as mean ± SD in Table 9.

The normality of the pH data was verified, and the homogeneity of variances was assessed using Levene’s test. As the assumption of variance homogeneity was met (p > 0.05), one-way analysis of variance (ANOVA) was applied to evaluate the potential differences in pH values between the analyzed conditions.

4. Conclusions

In summary, this comprehensive evaluation confirms that P. cattleyanum leaves are a multifunctional natural ingredient with significant value for skin health. The Cabo Verde leaf extract is rich in polyphenols (e.g., ellagitannins and flavonoids) that contribute to its potent antioxidant, anti-collagenase, anti-elastase, anti-tyrosinase, and antimicrobial activities (insert reference). Collectively, these bioactivities address major skin concerns, from photoaging (wrinkles and age spots) to acne and other infections, highlighting the broad dermatological potential of the extract.

Importantly, our findings bridge ethnopharmacological knowledge with modern science: many of P. cattleyanum traditional uses (e.g., treating wounds and inflammation) can be explained by the identified phytochemicals and their demonstrated effects (tissue-tightening and antimicrobial). This study is the first to thoroughly investigate P. cattleyanum from Cabo Verde, revealing that this underexplored plant population is a promising source of skin-active compounds. To fully harness its skin-protective properties, further research, including clinical trials and advanced formulation development, is warranted. Given the broad in vitro efficacy and lack of cytotoxicity of the extract, P. cattleyanum leaf extract emerges as an attractive candidate for incorporation into cosmeceutical products or adjunct herbal therapies aimed at skin rejuvenation and the treatment of skin disorders. This study provides a scientific foundation for developing natural skin health applications from P. cattleyanum and underscores the importance of conserving Cabo Verde’s medicinal flora to sustainably utilize this valuable resource.

Future research will focus on skin irritation testing, microbiological stability evaluation, and in vitro compound release studies to provide a more comprehensive assessment of the safety, quality, and functional performance of the developed topical formulation.