Physicochemical and In Vitro Biological Characterization of Usnea barbata Extract in Karanja Oil for Potential Applications in Skincare

Abstract

Plant extracts in vegetable oils are foundational and eco-responsible for skin care, combining their emollient properties with other additional benefits, derived from their antioxidant, antimicrobial and UV-absorbing activity. The present research conducted a complex investigation of Usnea barbata extract in Karanja oil (KO), aiming for its further incorporation into various cosmetic formulations. The lichen extract (UBKO) was obtained through cold maceration. Phytochemical screening was performed using the Folin–Ciocalteu method and Graphite Furnace Atomic Absorption Spectrophotometry (GFAAS). Physicochemical properties were evaluated via Fourier Transform Infrared Spectroscopy (FTIR) and Atomic Force Microscopy (AFM). The rheological behavior and oxidative stability of the oil samples, UBKO and KO, were also investigated. UBKO had a slightly lower density (0.827 vs. 0.955) and pH (4.22 vs. 4.86) than KO, and a slightly higher oxidative resistance, quantified as the induction period (IP) value (6.45 vs. 6.00). The total phenolic-equivalent content (TPC, µg GAE/mL oil sample) was significantly greater in UBKO than in KO (567.16 ± 14.96 vs. 433.26 ± 22.96, p = 0.001). The values of minimum inhibitory concentration (MIC, mg/mL) indicated significantly higher antibacterial effect against S. aureus and antifungal effect against C. albicans for UBKO than KO (9.62 ± 2.87 vs. 31.25 ± 18.75, p = 0.049, and, respectively, 5.06 ± 1.68 vs. 37.50 ± 12.50, p = 0.01). Finally, our results showed that UBKO had an estimated sun-protective factor (SPF) of 30.9, slightly higher than 29.8 for the base oil formulation, KO; these findings represent baseline in vitro UV-absorbing trends. All of these results suggest that U. barbata extract in Karanja oil may exhibit complementary bioactive properties with potential applications in skincare.

1. Introduction

Modern skincare science is increasingly oriented toward multi-active botanical formulations that deliver broad-spectrum efficacy while reducing reliance on synthetic chemical agents [1]. Natural products may exhibit complementary benefits when combined, making botanical extracts and vegetable oils attractive multifunctional ingredients in skincare formulations [2,3].

Karanja oil (KO) is a non-edible vegetable oil extracted from the seeds of the Pongamia pinnata (L.) Pierre, also known as Millettia pinnata (L.) Panigrahi, a tree native to India. Its main constituents are triglycerides and unsaturated fatty acids: oleic acid (44–71%), linoleic acid (10–18%), palmitic acid, and stearic acid [4]; it also contains three phenolic constituents (pongamol, karanjin, and cycloart-23-ene-3β,25-diol) with potential UVA and UVB sunscreen activities [5]. Karanja oil is described as a high-polarity active oil and is traditionally used for various chronic skin conditions [6]. Patents claim the use of Karanja oil in sunscreen formulations [7]. Dhule et al. incorporated Karanja oil into a herbal antifungal spray [8]. Karanjin, isolated from seed oil, can be used in cosmetic products such as soaps, body oils, and shampoos [9]. Recent studies report that its incorporation into modern formulations may benefit psoriasis and acne [10,11].

In the plant world, lichens are symbiotic organisms that produce specific, highly bioactive secondary metabolites. A widely studied lichen representative, Usnea barbata (L.) F.H. Wigg, belonging to the Parmeliaceae family, is available online as an extract at https://cosmileeurope.eu/inci/detail/16750/usnea-barbata-extract/, accessed on 10 June 2026. The selection of the lichen U. barbata as the active phytochemical solute was also based on both evolutionary and formulation considerations. It often thrives in extremely high-altitude environments with persistent exposure to intense solar ultraviolet radiation. Thus, U. barbata has evolved by overproducing specialized secondary metabolites, which act as natural, endogenous photoprotective screens and potent antioxidants [12,13]. Modern chemical analysis has identified numerous secondary metabolites from Usnea species [14]: usnic acid is the predominant bioactive constituent, followed by depsides and depsidones (barbatic acid, diffractaic acid, evernic acid) and phenolic acids [15]. Engel et al. demonstrated that U. barbata standardized extract can be a promising ingredient for the development of UV-protective skin care products or anti-inflammatory topical preparations [16].

Usnic acid was loaded into electrospun fibers and hydrogel membranes to optimize wound-healing efficiency [17,18]. Currently, it is used in various topical products as a fragrance, preservative, and antimicrobial agent, including deodorants, anti-acne treatments, creams, powders, toothpastes, mouthwashes, shampoos, and sunscreens. Its capacity to absorb UV light has attracted attention for photoprotection, with the (+)-enantiomer showing photoprotective and photostable properties similar to those of octocrylene in cosmetic formulations [19].

Our team analyzed the composition of U. barbata collected from the same unpolluted area in the Calimani Mountains at 900 m altitude, as documented in earlier studies. U. barbata extracts were prepared using various solvents—ethanol, methanol, ethyl acetate, and acetone—through different methods such as maceration and Soxhlet extraction. A comprehensive elemental analysis was conducted on dried lichen collected in 2020. Twenty-three metals were examined using inductively coupled plasma mass spectrometry (ICP-MS), revealing that native U. barbata contains all metals below the permissible limits for medicinal plants. Usnic acid (a dibenzofuran derivative) and other phenolic secondary metabolites, including phenolic acids (caffeic, p-coumaric acid, ellagic acid, chlorogenic acid, cinnamic acid, and gallic acid), were identified and quantified via UHPLC. Additionally, usnic acid was isolated from the dry U. barbata extract in ethyl acetate using semi-preparative chromatography. We have also extracted U. barbata in Canola oil, and a UHPLC method was validated for determining usnic acid content in U. barbata oil extract. The antimicrobial, antioxidant, and cytotoxic properties of various U. barbata extracts have been previously studied [20].

Both cosmetic ingredients (Karanja oil and U. barbata) are already used in skincare formulations. INCI decoder, the new INCI application (available online at https://incidecoder.com/, accessed on 10 June 2026), indicates that Karanja oil (used at 3–5% concentration) and U. barbata can serve as natural preservatives in cosmetic products, due to their antimicrobial activity. Additionally, the literature provides substantial evidence for antimicrobial, anti-inflammatory, antioxidant, and photoprotective activities—yet they act through distinct molecular mechanisms, exhibit different chemical polarities (lipophilic oil versus lichen-acid complex), and target complementary portions of the UV spectrum [16,21,22,23,24,25]. However, no literature reports the experimental approach of their combination.

In this context, we used Karanja oil (KO) to extract secondary metabolites from Usnea barbata, since vegetable oils are highly effective, eco-friendly solvents owing to their distinctive chemical and physical characteristics [26]. Then, we investigated the physicochemical and pharmacological properties of the oil lichen extract in KO (UBKO) compared with the extraction solvent. We hypothesized that dissolving the secondary metabolites of U. barbata (rich in lipophilic polyphenols and usnic acid) into pure KO would yield a complementary combination. The KO furanoflavonoids (karanjin and pongamol) serve as the primary optical shield, actively absorbing UVA/UVB photons. At the same time, the co-extracted lichen phenolics act as a secondary metabolic sink, systematically quenching the reactive oxygen species (ROS) that escape physical filtration.

Conventional products typically depend on synthetic organic UV filters like avobenzone, oxybenzone, or octocrylene, which face increasing restrictions from regulatory and health agencies due to their links to contact dermatitis, hormonal disruption, and environmental harm, such as coral reef bleaching [27,28,29,30,31,32,33,34,35,36,37,38] (Conventional Sunscreens: UV Filters & Research, available at https://spflist.com/conventional-sunscreens/ accessed on 10 June 2025). In contrast, our formulation utilizes a self-filtering, multi-targeted natural structure based on an active vehicle concept [39]. Instead of merely incorporating a lichen extract into an inert base, the extraction solvent (Karanja oil) serves as a broad-spectrum UV absorber [40]. It is thereby hypothesized to contribute as a preliminary in vitro UV-attenuating barrier, while concurrently suggesting a supportive, putative role in mitigating oxidative stress through an antioxidant cascade. However, it must be noted that these distinct conceptual mechanisms remain to be validated in advanced cellular or tissue-engineered skin models.

The novelty of our study lies in a complex physicochemical and pharmacological characterization of UBKO, a green extract with a specific vehicle–solute interaction, for potential incorporation into various cosmetic formulations.

2. Materials and Methods

2.1. Materials and Equipment

Karanja oil (KO), extracted from the seeds of Pongamia pinnata by cold pressing, was supplied by Fagron-Hellas (Trikala, Greece). The oil is highly pure and suitable for cosmetic uses. Its composition offers oxidative stability, a non-greasy feel, and good compatibility with human skin (Karanja seed oil, available at https://www.fao.org/4/x5043e/x5043e0e.htm, accessed on 9 June 2026).

In March 2024, U. barbata thalli were collected from the Călimani Mountains in Romania (47°28′ N, 25°13′ E at 900 m altitude). The freshly collected lichens were cleaned of debris and dried at 18–25 °C in a herbal room away from sunlight. For long-term preservation, dried samples were stored under similar conditions. The identification was performed by the Department of Pharmaceutical Botany at the Faculty of Pharmacy, Carol Davila University of Medicine and Pharmacy, using standard techniques. A voucher specimen is housed at the Herbarium of the Pharmacognosy Department, Faculty of Pharmacy, Carol Davila University of Medicine and Pharmacy (UBL 3/2024, Ph-UMFCD).

All chemicals, solvents, and reagents used were of high quality, ensuring reliable results. We sourced Dimethyl sulfoxide (DMSO), sodium carbonate (Na₂CO₃), and Folin–Ciocalteu reagent from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). For our experiments, we also used high-purity 69% nitric acid (trace grade) and prepared standard stock solutions containing 1000 mg/L H₃AsO₄ and Pb(NO₃)₂ in 0.5 M nitric acid. Additionally, we obtained 30% H₂O₂, 96% ethanol, methanol, crystal violet, acetic acid, and ultrapure deionized water from Merck Millipore (Burlington, MA, USA).

Microbial strains were acquired from the American Type Culture Collection (ATCC): Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Candida albicans ATCC 10231. Culture media came from two suppliers. Tryptic Soy Agar for bacteria was obtained from Sigma-Aldrich Merck (Dartmouth, Germany), while RPMI 1640 for fungi was supplied by American Biorganics (Buffalo, NY, USA).

2.2. U. barbata Extract in Karanja Oil

The oil lichen extract (UBKO) was prepared through 3-month cold maceration, as described earlier [25,41]. About 20 g of this material was precisely measured with a Kern analytical balance and placed in a 1000 mL brown glass container, to which 500 mL of KO was added (dried lichen/solvent ratio w/v = 1:25). The dried thallus of U. barbata was ground using a laboratory knife mill and standardized with a standard sieve. The particle size distribution was controlled by passing the material through successive sieves with 2.5 mm (DIN 1171) and 1.2 mm (DIN 117) mesh for homogenization [41].

Moisture content after drying: Prior to extraction, the lichen material was air-dried in a dark, well-ventilated room until it reached a constant weight. The residual moisture content was determined by loss-on-drying and was found to be below 5%, within the acceptable pharmaceutical limit to prevent accidental water-induced hydrolysis in the lipophilic medium.

The maceration was conducted in hermetically sealed, amber glass containers that were sterilized beforehand, stored in a dark, temperature-controlled environment (21–22 °C), manually shaken daily, and inspected periodically. No macroscopic or biochemical signs of spoilage were observed.

After this period, UBKO was filtered through cotton-mesh gauze into a brown vessel with a sealed lid and stored in a plant room away from sunlight.

Extraction yield (%) and efficiency evaluation: Since the extraction was performed directly into a lipid vehicle (Karanja oil) rather than a volatile organic solvent, the traditional mass-balance extraction yield (dry weight of extract/dry weight of plant) could not be directly determined by evaporation. Therefore, we appreciated the extraction efficiency by measuring the total phenolic content (TPC) transferred to the oil phase.

Control of microbial contamination during the 3-month maceration: To ensure microbiological safety during the extended 3-month maceration, multiple barriers were in place:

-

Pure Karanja oil is a lipophilic vehicle, with a thermodynamic state that fundamentally inhibits the growth and proliferation of bacteria, yeasts, and molds.

-

Both Karanja oil (due to karanjin) and Usnea barbata (due to usnic acid) possess well-documented, broad-spectrum antimicrobial and antifungal properties, acting as a self-preserving system.

The UBKO density (g/mL) was slightly lower than that of KO (0.827 < 0.955).

The pH values for KO and UBKO were measured with a pH meter (P601, CONSORT nv, Turnhout, Belgium). Equal volumes (1 mL) of UBKO and KO were transferred to 10 mL volumetric flasks, and 4 mL of deionized hot water (80 °C) was added. The mixtures were shaken vigorously for 5 min and then filtered. The pH value of the resulting aqueous extract was measured with the calibrated pH meter at 25 °C. UBKO has a lower pH value than KO (4.22 < 4.86).

2.3. Total Reducing Capacity (Folin–Ciocalteu Assay)

To establish a uniform system, 2 mL of each sample (KO and UBKO) was dissolved in 8 mL of polyethylene glycol 400 (PEG 400), ensuring consistent oil dispersion and enhanced solubilization of phenolic compounds in the reaction medium. Initial testing revealed that PEG 400 alone led to phase separation during the Folin–Ciocalteu assay, causing heterogeneous samples and unreliable spectrophotometric readings. Therefore, samples in PEG 400 were further diluted 1:1 (v/v) with DMSO. This PEG 400/DMSO co-solvent system improved sample miscibility and stability during analysis [42]. Briefly, 50 μL of each sample was combined with 50 μL of Folin–Ciocalteu (FC) reagent, 450 μL of distilled water, and 500 μL of 7% (w/v) Na₂CO₃ solution. The mixtures were mixed thoroughly and incubated in the dark for 50 min to allow the formation of the characteristic blue complex resulting from the reduction of the FC reagent by compounds present in the samples. Following incubation, samples were centrifuged at 10,000 rpm for 5 min to remove insoluble particles. Absorbance was measured at 765 nm using a UV–Vis FlexStation 3 spectrophotometer (Molecular Devices, San Jose, CA, USA).

This method showed good linearity within the concentration range of 25–250 µg/mL gallic acid (R² = 0.9965), with the calibration equation y = 0.0027x − 0.0261. The detection and quantification limits (LOD and LOQ) were 9.88 µg/mL and 29.94 µg/mL, respectively. All measurements were conducted in triplicate, with matrix interference controlled using DMSO and PEG 400/DMSO blanks. This approach allowed us to distinguish the solvent system’s contribution from that of the oil samples and to highlight PEG 400’s influence on the measured reducing capacity. Results are expressed as gallic acid equivalents (GAE) per mL or per g, representing the total phenolic-equivalent content (TPC). For results in μg GAE/g, the conversion from μg GAE/mL was based on the system’s density. The assay used PEG 400 alone as a control to clearly illustrate the oils’ composition [41].

2.4. FTIR Spectra

FTIR spectra were obtained using an FTIR spectrometer (FT/IR-4200 apparatus, from JASCO, Tokyo, Japan). The spectral experiments were obtained in the wavelength range between 4000 and 400 cm⁻¹, with a 4 cm⁻¹ resolution. All spectra were recorded at ambient temperature and are presented in transmittance values [41].

2.5. AFM Analysis

For Atomic Force Microscopy (AFM) analysis, KO and UBKO (20 µL each) were diluted in 2 mL of 96% ethanol. Then, the samples were further deposited on a clean glass substrate and heated at 20 °C for 30 min. This additional step aimed to ensure solvent evaporation and proper sample adhesion. AFM analysis was performed in enhanced contrast mode [41]. The resulting line scans illustrated the surface profiles of both studied samples.

2.6. Heavy Metals Content

Heavy metals, arsenic (As) and lead (Pb), were detected using Graphite Furnace Atomic Absorption Spectrophotometry (GFAAS) as previously described. The measurements were carried out with a SOLAAR 6M (Thermo Electron Inc., Waltham, MA, USA) atomic absorption spectrometer equipped with a deuterium lamp for background correction. The specific wavelengths used were 193.7 nm for As and 283.3 nm for Pb. Concentrations of both metals in each oil sample were determined based on their respective regression lines, which relate absorbance to concentration. All measurements were performed in duplicate [41].

2.7. Rheological Measurements

Rheological measurements were conducted using a B One Plus rotational viscometer (Lamy Rheology, Champagne-au-Mont-d’Or, France), which offers a full-scale accuracy of ±1% and a repeatability of 2%. The instrument is equipped with seven spindles (RV1–RV7), each designed to operate within a defined viscosity range. For the present study, spindle RV3 was selected and immersed in 50 mL of each oil sample for viscosity determination. Measurements were carried out at rotational speeds of 50, 100, 150, 200, and 250 rpm, with each reading recorded after 10 s of stabilization. All analyses were performed under controlled temperature conditions of 22 °C [41]. The spreading properties of the samples were evaluated using an extensometer (Epsilon Technology Corp., Jackson, WY, USA). A fixed volume of 0.5 g of oil was carefully deposited at the center of the lower glass plate, after which a second glass plate (50 g) was placed on top to initiate spreading. The system was subsequently subjected to incremental loading by adding weights of 50, 100, 200, and 500 g. After a 1-min relaxation period for each applied load, the diameter of the spread sample was recorded. The area of the spreading was then calculated using the formula πr².

2.8. Oxidative Stability

The Velp OXITEST reactor (Velp Scientifica Srl, Usmate Velate, MB, Italy), which speeds up oxidation under carefully controlled and consistent conditions, was used to evaluate the oxidative stability of UBKO and KO by measuring the induction period [43], as previously described [41].

2.9. Antioxidant Activity

2.9.1. DPPH Assay

We evaluated the radical-scavenging ability of UBKO using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, as previously described [44]. The DPPH method relies on the discoloration of a solution containing the DPPH free radical (Sigma-Aldrich Inc., Burlington, MA, USA) when it interacts with antioxidants in the tested sample. For each sample, multiple dilutions in methanol were prepared (1:1, 1:5, 1:10, and 1:25) to examine the dose-dependent variability of the antioxidant effect.

Each sample was combined with the DPPH working solution at a 1:2 ratio, starting with an optical density (OD) value of around 0.8.

We performed an assessment by measuring the decrease in OD at 517 nm over one hour. Additionally, we conducted endpoint measurements at 5, 30, and 60 min to analyze the decrease in OD:

ΔOD (%) = 100 × (OD_DPPH − OD_{DPPH + sample})/OD_DPPH

where OD_DPPH corresponds to methanol + DPPH working solution. This was used to demonstrate the antioxidant effect of the tested oil samples.

2.9.2. ABTS Assay

The 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) method assesses a sample’s capacity to neutralize the ABTS+∙ free radical. The stock solution, containing 7 mM ABTS and 2.45 mM K₂S₂O₈, was stored in the dark at 4 °C for 16 h to produce the green ABTS+∙ radical. This was diluted with distilled water to a final optical density of 0.8 at 714 nm, creating the working solution. Oil samples, both undiluted and diluted at ratios of 1:1, 1:5, 1:10, and 1:25, were mixed with the working solution at a 1:3 ratio, and their absorbance was measured at 714 nm after 10 min. A control sample was prepared using ethanol instead of the oil. The results are expressed as the percentage decrease in optical density (% ΔOD) [44].

ΔOD (%) = 100 × (OD_blank − OD_sample)/OD_blank

This was considered to express the antioxidant effect of the tested oil samples [44].

2.10. Antibacterial and Antifungal Activity

To assess the antimicrobial activity of KO and UBKO against the most common pathogens (S. aureus ATCC 25923, E. coli ATCC 25922, and C. albicans ATCC 10231), we first determined their minimum inhibitory concentration (MIC). Subsequently, using sub-MICs (MIC/2 and MIC/4), we evaluated the oil samples’ ability to inhibit microbial adhesion to an inert substrate (AC, expressed as a percentage) [45].

2.10.1. Assessment of the Minimum Inhibitory Concentrations

To assess the inhibitory activity of the oil samples, a 20% Tween 80 (T80) solution in ethanol was used to dissolve the bioactive compounds, facilitating their dispersion in the aqueous culture medium. All microbiological assays included vehicle control wells using the Tween 80–ethanol system [45].

Suspensions with an optical density of 0.5 McFarland for bacteria and 1 McFarland for fungi were prepared. The following procedures were carried out: 100 μL of TSA or RPMI 1640 was distributed into each of the 96-well plates, followed by 100 μL of a 100 mg/mL solution of the compounds under investigation in the first well. A binary serial dilution scheme was then performed up to the tenth well. Subsequently, 20 μL of suspension from the strains designated for analysis was added, ranging from well 1 to well 12. Well 11 was with vehicle control (T80), while well 12 was designated for negative control. Absorbance measurements were conducted at 620 nm utilizing a Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) [44].

2.10.2. Assessment of How Oil Samples Affect Microbial Adherence to the Inert Substrate

The impact of UBKO and KO on biofilm formation was examined using the method previously outlined [45]. The adhesion level of the tested strains to an inert substrate was measured at sub-MICs (MIC/2 and MIC/4) after fixing with methanol and staining with a 1% crystal violet solution in 33% acetic acid [45].

The adherence capacity (AC%) of the microbial strains was ascertained through the utilization of the subsequent formula.

AC (%) = 100 × (A_sample − A_blank)/(A_control − A_blank)

where A_sample is the absorbance at 490 nm of the tested oils, while Acontrol represents the absorbance at 490 nm of the negative control, represented by untreated microbial strains [44].

2.11. Sunscreen Properties

Volumes of 1 mL of the studied oil sample were transferred to a volumetric flask (100 mL) and diluted with ethanol to volume. The samples were then ultrasonicated for 5 min. The SPF values of the oil samples were measured using a spectrophotometer, Perkin-Elmer Lambda 35 (Perkin-Elmer Inc., Waltham, MA, USA). The experiments were made in transmission mode. The samples were placed in microcuvettes with a 10 mm light path, and the absorption values were recorded over the range of 290 to 320 nm, at 5 nm intervals. Each measurement was performed three times, and the average value was used. SPF values were calculated using the Mansur equation [44] (Equation (1)).

$$SPF=CF\times {\displaystyle {\sum }_{320}^{290}}EE\left(\lambda \right)\times I\left(\lambda \right)\times Abs\left(\lambda \right)$$

(1)

where CF = correction factor (10), I(λ) = intensity of solar light at wavelength λ, EE(λ) = erythemogenic effect of radiation at wavelength λ, and Abs (λ) = absorbance of wavelength λ by the preparation solution. The value of $EE\left(\lambda \right)\times I\left(\lambda \right)$ is constant.

2.12. Statistical Analysis

Most measurements were carried out three times to ensure consistent results, and the findings are presented as averages with their standard deviations. We analyzed the data using SigmaXL v.11.03/2025 (SigmaXL Inc., Kitchener, Ontario, Canada) and Microsoft Excel v.16.0 19328 (Microsoft Corporation, Redmond, WA, USA). To identify significant differences among variables (p < 0.05), we used One-Way ANOVA and Means Matrix, 2-Sample t-test, and Kruskal–Wallis test.

3. Results

3.1. Total Reducing Capacity (Folin Ciocalteu Assay)

The results obtained by the Folin–Ciocalteu assay (

Table 1. Total reducing capacity of both oil samples, expressed as total phenolic-equivalent content (µg GAE/mL oil or µg GAE/g).

Sample	Total Phenolic-Equivalent Content (TPC)
	µg GAE/mL Oil + PEG 400	µg GAE/mL Oil Sample	µg GAE/g Oil + PEG 400	µg GAE/g Oil Sample
KO	693.75 ± 22.96 a	433.26 ± 22.96 a	652.61 ± 20.28 a	407.57 ± 20.28 a
UBKO	827.66 ± 14.96 b	567.16 ± 14.96 b	773.55 ± 13.98 b	530.08 ± 13.98 b
PEG 400	260.49 ± 0.27 c	-	229.23 ± 0.24 c	-

KO—Karanja oil; UBKO—U. Barbata extract in Karanja oil; PEG 400—polyethylene glycol 400. In the same column, the values marked with different superscripts are significantly different. The TPC values reported for “oil + PEG 400” correspond to the complete emulsified system corrected only against the DMSO blank, whereas the “oil sample” values represent TPC values further corrected by subtraction of the PEG 400/DMSO blank contribution. The statistical analysis was performed using one-way ANOVA for correlation with PEG400 and an unpaired t-test with Welch’s correction for oil comparisons (p < 0.05). The normality and homogeneity of variances tests confirmed the appropriateness of using parametric methods.

) show significant differences in the total phenolic-equivalent content (TPC, µg GAE/mL) between the KO and UBKO. Karanja oil had an appreciable capacity to reduce FC reagent; U. barbata extraction in KO significantly increased the total reducing capacity (567.16 vs. 433.26 µg GAE/mL in the oil sample; p = 0.001; Table 1). The oil samples emulsified with PEG 400 had a higher capacity to reduce FC reagent, with significant differences between KO and UBKO, because the Folin–Ciocalteu assay is based on an electron-transfer (redox) mechanism and exhibits known interferences with non-phenolic reducing compounds (Table 1).

The positive value obtained for the PEG 400 control (260.49 ± 0.27 µg GAE/mL) was due to the reducing capacity of the terminal hydroxyl groups of the short polymer, enhanced by possible traces of aldehyde impurities or reducing sugars from the raw material, typical of low-molecular-weight materials [46]. Therefore, the results provide an estimate of the total reducing capacity of the oil samples (KO and UBKO). Since the method also responds to other reducing compounds, the obtained values do not represent a specific measurement of phenolic secondary metabolites’ concentration.

3.2. FTIR Analysis

The IR spectrum of KO reveals molecular structures rich in saturated aliphatic chains, evidenced by C–H stretching vibrations at 2922.6 cm⁻¹ and 2853.2 cm⁻¹, which are typical of methylene (–CH2) groups in alkane derivatives (Figure 1, blue line) [47]. The C–H bending vibrations are observed at 1463.7 cm⁻¹ and 1376.0 cm⁻¹, confirming the presence of alkyl chains. The absorption at 722.2 cm⁻¹ corresponds to the rocking vibration of methylene groups, supporting the existence of long-chain aliphatic backbones. The weak band at 3006.5 cm⁻¹ indicates a =C–H stretching vibration, but the high transmittance suggests a low proportion of double carbon–carbon bonds compared to the saturated (single-bonded) carbon atoms in the oil components (Figure 1, blue line). The strong band at 1743.3 cm⁻¹ confirms the presence of ester groups assigned to the carbonyl (–C=O) stretch vibration. The bands observed at 1230.4 cm⁻¹, 1162.9 cm⁻¹, and 1116.6 cm⁻¹ are assigned to the C–O stretching vibrations. The free acids are below the detection limit, as shown by the absence of the characteristic hydroxyl-stretching broad O–H band from the range of 3500 to 2500 cm⁻¹. The IR spectrum suggests that triglycerides may be the most abundant components (Figure 1, blue line). The IR spectrum of UBKO shows some differences in the 1600–400 cm⁻¹ region, but the spectral shapes are similar (Figure 1, red line). These differences in the fingerprint region suggest slight structural alterations, such as changes in the chemical environment, cation substitution, or various lattice vibrations/degrees of ordering between the two oil samples, thereby maintaining a pattern similar to the KO. This similarity, together with the absence of significant band shifts or new absorption bands, demonstrates that U. barbata metabolites extracted in KO do not undergo chemical interactions, but only physical associations.

3.3. AFM Analysis

The AFM images of KO and UBKO are shown in Figure 2. Figure 2a displays the scan over a (10 × 10) µm² area for KO, while Figure 2b shows a similar scan for UBKO. Figure 3c,d present scans over a (4 × 4) µm² area for KO and UBKO, respectively. The characteristic line-scan profiles are indicated at the position marked by the red line. Additionally, Figure 2e–h show histograms of the peak-to-valley (Rpv) and roughness (Rq) parameters.

AFM analysis shown in Figure 2 highlights several morphological differences between KO and UBKO at both examined scales of (10 × 10) µm² and (4 × 4) µm². KO exhibits a mostly uniform surface, characterized by larger structures or aggregates that are sporadically dispersed. This suggests the presence of microdomains on the surface, leading to a vertical red line scale variation of approximately 17 nm (from −5 to 12 nm), specific to its lipid composition (Figure 2a). In contrast, UBKO displays a much more heterogeneous surface, characterized by smaller structures that are more numerous and evenly distributed (Figure 2b). This results in lower variations of around 9 nm (from −3 to 6), as indicated by the red line scan in Figure 2b. These formations suggest changes in the surface morphology and structural organization of the oil matrix following extract incorporation. The AFM topography of both compounds is also confirmed at a smaller scale (4 × 4) µm², where the morphological details become more evident (Figure 2c,d).

The analyzed roughness parameters, Rpv (peak-to-valley parameter) and RMS (root mean square) roughness (Rq), respectively, exhibit lower values in the case of KO despite the presence of larger aggregates. This observation confirms its smooth and homogeneous nature (Figure 2e,f). The KO sample is characterized by an Rq of 5.9 nm and an Rpv of approximately 98.1 nm across the entire scanned area of (10 × 10) µm² (Figure 2e). The relatively low density of KO (0.9548 g/cm³) indicates a less dense molecular association, typical of lipid systems. This KO structure facilitates molecular mobility, thereby supporting the formation of larger, yet sporadically distributed, aggregates that contribute to a relatively smooth surface.

In contrast, the U. barbata phytochemicals extracted in Karanja oil resulted in much higher roughness parameter values across the entire scanned area and along the corresponding red line profile (Figure 2e–h). This indicates a noticeable increase in the roughness and local height variations. UBKO is characterized by an Rq of about 9.3 nm and a Rpv of about 166.6 nm. Lichen constituents can induce surface reorganization, possibly by forming smaller nanoscale structures, thereby increasing surface roughness.

3.4. Heavy Metals Content

The arsenic content (μg/g) is higher in UBKO compared to KO (0.203 versus 0.131), while the lead content is lower (0.056 versus 0.065). The data are presented in both μg/L and μg/g in

Table 2. The As and Pb content in KO and UBKO.

Metal	Oil Sample	Sample Weight	Heavy Metal Concentration
		G	μg/L	μg/g
As	KO	0.315	4.122	0.131
	UBKO	0.318	6.450	0.203
Pb	KO	0.315	2.049	0.065
	UBKO	0.318	1.788	0.056

KO—Karanja oil; UBKO—U. barbata extract in Karanja oil.

. The arsenic levels in both oil samples stay within the range considered safe for cosmetic or unrefined vegetable oils. These findings align with recent literature, which reports arsenic concentrations in vegetable oils typically between 0.05 and 0.20 mg/kg. Both oil samples exhibit similar lead levels (0.065 and 0.056 μg/g), which are well below the maximum permissible limit of 0.100 μg/g established by Regulation (EC) No. 1881/2006 for lead in vegetable oils and fats [48].

3.5. Rheological Properties

The rheological curves of KO and UBKO, along with the linear fit equations for shear stress, are shown in Figure 3. We examined the rheological behavior of KO by looking at how shear stress (mPa) relates to shear rate (rpm) (see Figure 3a–c). A clear linear relationship was found across the explored range, which suggests Newtonian behavior—meaning shear stress increases directly with shear rate.

The experimental data were fitted using a linear regression model of the form (Equation (2))

$$y=a+b\mathrm{x}$$

(2)

where y is the shear stress (τ), x is the shear rate ($\dot{\gamma }$), and slope (b) represents the dynamic viscosity (η), according to Equation (3) [49].

$$\eta ={\displaystyle \frac{\tau }{\dot{\gamma }}}$$

(3)

The high correlation coefficient (r² ≈ 0.999) confirms a strong linear dependence between shear stress and shear rate. Although a rapid decrease in dynamic viscosity was observed with increasing or decreasing shear rate, in the initial range of 50 to 100 rpm (at low shear rates), the KO exhibits shear-thinning behavior. Subsequently, the dynamic viscosity ranges from approximately 250 to 260 mPa·s at higher shear rates, indicating a transition to Newtonian behavior. This variation can be attributed to the low torque values (0.115–0.485 mN·m) recorded during measurements. Additionally, the composition of higher fatty acids influences KO’s shear-thinning behavior, leading to non-Newtonian behavior at lower shear rates due to molecular interactions involving monounsaturated fatty acids, which result in higher dynamic viscosity at low shear rates. Therefore, the linear shear stress–shear rate relationship is considered a more reliable parameter for determining KO’s fluid behavior. Overall, these results demonstrate that the analyzed KO sample behaves as a Newtonian fluid within the tested shear rate range.

The rheological behavior of UBKO was assessed by analyzing the relationship between shear stress and shear rate (Figure 3d). A linear correlation was observed throughout the entire tested shear rate range, and the dynamic viscosity remained mostly constant between 100 and 250 shear rates, confirming that the UBKO also displays Newtonian behavior, similar to KO. Linear regression was conducted using the same model as before (Equation (2)). Additionally, the high correlation coefficient (r² ≈ 0.997) supports the strong linear relationship between shear stress and shear rate and indicates no yield stress. The nearly constant dynamic viscosity suggests that UBKO’s flow resistance is independent of shear rate, a hallmark of Newtonian fluids.

Initially, when the upper plate was applied, the stretching surface of UBKO was smaller than that of KO, indicating its spreadability (Figure 4).

Later, when more weights are added, UBKO’s extensibility remains lower than KO’s (7800.88 ± 1851.54 compared to 9727.16 ± 2723.88, p > 0.05; Figure 4). The spreadability of the samples correlates with their viscosity behavior, suggesting that KO will likely spread quickly and evenly over the skin without needing much pressure. Additionally, extensibility indicates KO’s behavior within various fluid or semi-solid systems. The results show that KO can spread uniformly across different carriers with minimal energy input. Formulation cohesiveness influences extensibility, and KO maintains its structural integrity and flexibility. Gobi et al. demonstrated that viscoelastic properties significantly impact adhesion [50].

A robust link between spreading and viscoelasticity is observed in UBKO, much like in KO. Nonetheless, the minor variations observed imply that lichen components form new interparticle bonds within the oily system, as reflected in the changes in rheological properties.

3.6. Oxidative Stability

Oxidative stability was quantitatively assessed by the induction period (IP), recorded in hours. This is the time point where the tangent lines drawn before and after the inflection point intersect. The IP represents how long it takes for oxidation to initiate, potentially resulting in noticeable rancidity or a sharp rise in oxidation rate.

Generally, a longer IP correlates with higher oxidation resistance and shelf life [43]. Our results show that UBKO had slightly greater oxidative stability (IP = 6.45) than KO (IP = 6).

3.7. Antioxidant Activity

To assess the antioxidant activity of KO and UBKO accurately, samples were diluted with methanol at ratios of 1:1, 1:5, 1:10, and 1:25. These were then compared to a solvent-based standard to analyze their dose–response behavior.

The in vitro radical-scavenging ability of KO and UBKO, evaluated with the DPPH method, was determined using both kinetic and endpoint assays (Figure 5a).

The KO sample stock solution reported the strongest DPPH radical-scavenging activity (ΔOD = 70.92%), higher than UBKO (ΔOD = 64.64%). UBKO dilutions exhibited greater antioxidant capacity than KO, which generally decreased with dilution grade from 1:1 to 1:25 (Figure 5a).

Results from the ABTS method are shown in Figure 5b. We were unable to assess the KO and UBKO undiluted samples or the lowest dilutions because an emulsification process rendered them unsuitable for reading, as the ABTS working solution was prepared with water as the solvent, which is incompatible with the hydrophilic antioxidants in the evaluated samples. Therefore, results were presented alongside the dynamics of the ABTS solution over the same time span (Figure 5b). The evaluated dilutions ranged from 1:5 to 1:25. The radical-scavenging activity of both oil samples increased over time, as indicated by decreases in OD values (Figure 5b).

The results revealed that KO has antioxidant capacity, with statistically significant differences among the tested dilutions. UBKO also exhibited antiradical activity, though variation across the tested dilutions was less pronounced. Overall, both samples showed reasonable free radical-scavenging activity over the 1:5 to 1:25 dilution range.

3.8. Antibacterial and Antifungal Activity

The antimicrobial activity assessment targeted two aspects: (1) establishing the minimum inhibitory concentration (MIC) and (2) assessing KO and UBKO’s capacity to prevent bacterial and fungal biofilm formation.

To assess the inhibitory activity of the oil samples, a 20% Tween 80 (T80) solution in ethanol was employed for solubilization, facilitating the dispersion of bioactive compounds in the aqueous culture medium. Tween 80 also served as a positive control in the microbiological assays.

All determined MIC values (mg/mL) shown in

Table 3. MIC values of oil samples and solvent control.

Microbial Cell Line	UBKO	KO	T80
	MIC (mg/mL)
S. aureus ATCC 25923	9.62 ± 2.87 a	31.25 ± 18.75 b	50 ± 0.00
E.coli ATCC 25922	50 ± 0.00	37.50 ± 12.50	37.5 ± 12.50
C. albicans ATCC 10231	5.06 ± 1.68 a	37.50 ± 12.50 b	50 ± 0.00

KO—Karanja oil; UBKO—U. barbata extract in Karanja oil, T80—Tween 80. In the same row, the values marked with different superscripts are significantly different (p < 0.05).

were considerably higher than those of conventional antibiotics’ values (mg/mL) reported by Rankovic et al. for the same ATC bacterial and fungal lines [51]. UBKO had the highest inhibitory activity against S. aureus and C. albicans, and the lowest antibacterial efficacy against E. coli (Table 3).

The influence of KO and UBKO at sub-MICs (MIC/2 and MIC/4) on microbial adherence compared to the negative control (untreated microorganisms) is illustrated in Figure 6.

The results show that UBKO inhibited the adherence of all bacterial and fungal strains at both sub-MICs tested, and the effect was directly proportional to the concentration tested. The AC values for all pathogens were significantly lower than those of negative controls (p < 0.05, Figure 6a). The highest antiadherence capacity was observed against C. albicans, with very little difference in AC (%) values at both sub-MICs (MIC/2 vs. MIC/4, 7.89 vs. 8.49; Figure 6a). Higher AC values were recorded on S. aureus (31.99 vs. 26.62, Figure 6a). UBKO at MIC/2 showed an E. coli adherence inhibition almost 6 times higher than MIC/4 (6.19 vs. 36.77, Figure 6a).

Contrariwise, KO at MIC/4 stimulated biofilm formation for all pathogens; the highest adherence capacity was observed in E. coli, followed by S. aureus and C. albicans (294.52 vs. 167.91 and 119.33, Figure 6b). At MIC/2, KO considerably inhibited the adherence of C. albicans and S. aureus (46.55% and 72.24%, respectively), while its stimulatory effect on E. coli remained high and recorded a very low diminution (AC = 260.30 at MIC/2 vs. 294.52 at MIC/4, Figure 6b).

3.9. UV Absorption

SPF values serve as a standard for evaluating the efficacy of sunscreen formulations against UV-B radiation. SPF value represents the ratio of UV energy required to achieve a minimum erythematous dose (MED) in protected skin relative to unprotected skin. The absorbance values of the KO and UBKO samples obtained using the UV spectrophotometric method are shown in

Table 4. The results for KO and UBPO absorption using the spectrophotometric method.

Wavelength (cm−1)	EE(λ) × I(λ)	Absorbance
		KO	UBKO
290	0.0150	2.80294	2.96479
295	0.0817	2.92969	3.06945
300	0.2874	2.98885	3.11607
305	0.3278	3.00255	3.10471
310	0.1864	2.97773	3.09127
315	0.0837	2.96323	3.03929
320	0.0180	2.91667	3.01506

KO—Karanja oil; UBKO—U. barbata extract in Karanja oil. EE(λ) represents the erythemogenic effect of radiation at wavelength λ; I(λ) indicates the solar light intensity at that wavelength; Abs(λ) denotes the absorbance of wavelength λ by the preparation solution.

.

The SPF value of KO is 29.8, and for UBKO is 30.9. It was observed that extracting U. barbata bioactive constituents into Karanja oil increased the SPF by approximately 1 unit. These results show that U. barbata extraction did not significantly change the estimated SPF value under the tested conditions. However, the formulation kept UV-absorbent and antioxidant properties that could be important for photoprotective uses. The results suggest that the KO and UBKO can be considered for inclusion in the sunscreen formulations, together with other ingredients.

4. Discussion

Our study proposes a novel combination of two known bioactive natural products (U. barbata extract in Karanja oil) with potential benefits in skincare. The research had a broad experimental scope, integrating physicochemical and biological characterization relevant to cosmetic applications.

The selection of a 3-month maceration period at room temperature for UBKO preparation was determined by the unique mass-transfer kinetics present in a highly viscous lipid medium. According to fundamental diffusion principles, as modeled by the Stokes–Einstein relation, the molecular diffusion coefficient of a solute is inversely proportional to the dynamic viscosity of the extraction solvent. While volatile organic phase extractions using low-viscosity media such as acetone or ethanol reach equilibrium within hours, unrefined vegetable oils are composed of bulky, long-chain fatty acid triglycerides with dynamic viscosities nearly two orders of magnitude higher at 20 °C [52]. As a result, the mass transfer of lipophilic phytocompounds across the dense, fibrous cortical and medullary layers of the U. barbata thallus is structurally limited. To ensure complete exhaustion of the vegetal matrix without using thermal acceleration—which would compromise the photostability of the furanoflavonoids and cause oxidative degradation of the oil’s unsaturated lipid profile—the extraction period was extended to 3 months [25,53]. This kinetic compensation ensured maximum passive solute saturation while preserving the intact biological functionality of the photoprotective constituents.

Both oil samples (UBKO and KO) were investigated concomitantly to compare the properties of U. barbata oil extract with those of Karanja oil. Our previous research on U. barbata underlines the complex design of the present study. Phenolic-equivalent content accounts for all bioactive constituents of U. barbata and Karanja oil, with or without phenolic structure. In KOs extracted from Pongamia tree seeds harvested from two different zones in India, Watti et al. reported 13.5–15.2 mg GAE/g [54]. Our results indicate lower TPC levels in both KO (407.57 µg GAE/g) and UBKO (530.08 µg GAE/g), probably due to differences in the KO origin and preparation. However, it must be acknowledged that the chemical standardization of the UBKO remains our priority in further studies. Precisely identifying bioactive components using high-performance liquid chromatography (HPLC) or gas chromatography–mass spectrometry (GC-MS) is an important direction for future work.

The potential complementary effects of the formulation components arise from the possible physical association between the phenolic compounds in the extract and the bioactive constituents of KO, which, together, could enhance UBKO’s pharmacological potential. One of the targeted screenings was focused on the most critical toxic elements rather than a comprehensive multi-element analysis. Arsenic (As) and lead (Pb) were chosen as representatives of heavy metals because of their toxicological significance, regulatory importance, and known affinity for plant- and lichen-based matrices. Previous analysis of dry lichen revealed a Pb concentration of 1.296 µg/g, with arsenic undetected. The heavy metal levels in UBKO and KO are below the maximum permissible limits, indicating adherence to European safety standards. These findings align with recent literature, which reports typical Pb levels in vegetable oils ranging from 10 to 80 µg/kg. This supports the method’s validity and excludes external contamination in both oil samples. The HNO₃/H₂O₂ mineralization technique using the Ethos Easy microwave system, combined with GFAAS detection, proved effective, reproducible, and sensitive for trace heavy metal measurement in vegetable oils. The WHO considers arsenic and lead priority contaminants due to their cumulative, carcinogenic, and neurotoxic effects and emphasizes the absence of a safe exposure level, especially for lead. Lichens like U. barbata are well-known bioaccumulators of atmospheric pollutants, with arsenic and lead being among the most frequently reported metals. From an analytical standpoint, both elements can be transferred into lipid matrices and precisely measured at trace levels using GF-AAS. In contrast, metals such as Fe, Zn, and Cu are either essential, naturally abundant, or less relevant to safety in this context.

Accelerated oxidative stability tests were conducted under extreme thermal stress (at 90 °C) to induce rapid degradation. According to fundamental lipid oxidation kinetics (the Arrhenius equation and Van’t Hoff’s rule), the rate of lipid auto-oxidation approximately doubles for every 10 °C increase in temperature [55]. Consequently, a 7.5% relative increase in the IP at an elevated temperature translates into an exponential extension of shelf life by several months under ambient storage conditions (20–25 °C) [56]. Because the rate of triglyceride auto-oxidation decreases exponentially as temperature approaches ambient storage levels (20–25 °C), this 7.5% relative improvement in thermal oxidative resistance translates to a substantial extension of the product’s commercial shelf life, effectively delaying rancidity and the formation of secondary malodorous decomposition products [57] Regarding the oxidative stability of the formulations, the pure KO vehicle showed a baseline Induction Period (IP) of 6.00 h, which increased to 6.45 h with the addition of U. barbata metabolites. Although this absolute increase of 0.45 h is modest under accelerated, high-temperature testing conditions (90 °C), its practical and commercial significance is reasonable when viewed in terms of lipid degradation kinetics.

This baseline stabilization also has significant in-use relevance. When applied as a topical sunscreen film, the unsaturated fatty acids of KO are susceptible to immediate photo-oxidation under direct sunlight. The dissolved polyphenols and (+)-usnic acid from U. barbata serve as efficient, sacrificial, chain-breaking antioxidants. By trapping alkylperoxyl radicals, the lichen metabolites preserve the structural integrity of the furanoflavonoid carrier during the critical hours of skin sun exposure. Importantly, this preservation is achieved without the addition of controversial synthetic stabilizers such as butylated hydroxytoluene (BHT) or butylated hydroxyanisole (BHA), maintaining the entirely natural, eco-friendly, and non-irritating profile of the photoprotective system.

Excessive UV radiation can promote the growth of stress-resistant microorganisms [58,59,60]. Therefore, the antimicrobial activity of KO and UBKO was tested against common pathogens, including S. aureus, E. coli, and C. albicans. Our findings showed that UBKO exhibits moderate and dose-dependent antimicrobial and antibiofilm effects against S. aureus and C. albicans. These results align with the existing literature, which confirms its antibacterial activity, especially against Gram-positive bacteria [24,51,61,62,63,64]. In UBKO, usnic acid is an antibacterial agent against Gram-positive bacteria, with its dibenzofuran structure disrupting RNA and DNA synthesis and inhibiting DNA replication and elongation [65]. At this stage, these preliminary antimicrobial properties should be regarded only as a supporting characteristic that may contribute to the overall microbiological safety and secondary protection of the further topical formulations.

Karanja oil-specific phenolic constituents (karanjin, pongamol, and cycloart-23-ene-3β,25-diol) act through a different route—their bactericidal activity is primarily due to inhibition of bacterial cell membrane synthesis [22]. The antibacterial and antifungal activities of Pongame Oiltree seeds are well documented in the literature. Additional studies confirmed inhibitory effects on S. aureus, E. coli, and C. albicans [66,67,68,69]. Devidas et al. reported that unsaturated fatty acids (linoleic, oleic, linolenic) in the oil also independently contribute to antimicrobial activity through membrane-disruptive mechanisms [9]. Thus, the UBKO anti-staphylococcal and anti-candida efficacies are significantly higher than those of KO. Moreover, the KO biofilm-formation stimulation across all pathogens at MIC/4 can be explained. All our results suggest a potential complementary effect of triterpenoids and flavonoids, which could represent a new approach for treating bacterial infections [70].

Phenolic metabolites and unsaturated fatty acids also contribute to the antioxidant and sunscreen properties of UBKO and KO. UBKO’s higher TPC, derived from lichen secondary metabolites, gives it higher DPPH-radical scavenging activity, better oxidative stability, and a greater photoprotective effect than KO.

KO shows higher activity in the stock solution. The apparent discrepancy between the DPPH and ABTS assays may be due to their slightly different solvent affinities. The DPPH assay is better suited for measuring lipophilic antioxidant compounds, while the ABTS assay can detect both hydrophilic and lipophilic antioxidant activity. Since KO is an oil-based sample, its higher antioxidant activity in the undiluted sample may be more effectively measured using the DPPH method. Additionally, the scavenging activity in both assays increased with concentration, confirming the dose-dependent antioxidant potential of the tested oils. We did not use any standard, as we compared our results with those obtained with the specific radical scavenging reagents. Nevertheless, a comparative evaluation of KO and UBKO under identical experimental conditions still provides relevant information on their relative free radical scavenging capacities. We acknowledge the inherent limitations of the ABTS assay for evaluating antioxidant activity in oil matrices, as solvent polarity and matrix effects may influence radical-scavenging measurements. Furthermore, the absence of a standard antioxidant reference precludes direct comparison of antioxidant capacity with established benchmark compounds. Consequently, the ABTS and DPPH results are considered comparative measurements obtained under the applied experimental conditions rather than absolute indicators of antioxidant potential. Accordingly, the interpretation of the antioxidant behavior of KO and UBKO is based solely on the obtained results.

Before in vivo testing in humans, in vitro testing can be used as a formulation tool to identify novel filters, optimize combinations of existing ones, and prescreen protective formulas [71]. The aim of our study was to determine whether UBKO can be considered a bioactive ingredient in green cosmetic formulations, which are increasingly desired in modern society. The Mansur method used in the present study is an in vitro spectrophotometric technique based on a dilute solution [72]. It mathematically demonstrates the chemical filtering capacity of UV radiation; however, it does not simulate the behavior on viable human skin, such as film adhesion or photostability, nor does it account for cellular biological mechanisms. Nevertheless, it is a method on which the standard protocol is based. The prevailing international standard is ISO 24443:2021, a standardized testing method used in the cosmetics industry to evaluate the in vitro UVA protection provided by sunscreen products. Currently, ISO 24443:2021 is one of the most widely used in vitro methods for determining sunscreen UVA photoprotection, available online on https://www.iso.org/obp/ui/en/#iso:std:iso:24443:ed-2:v2:en, accessed on 10 June 2026. A slight increase in the estimated SPF value of UBKO compared with the KO sample using the Mansur spectrophotometric assay was observed. Still, the Mansur method provides only a preliminary estimation of UVB protection based on spectrophotometric measurements [73,74,75]. Given their baseline spectrophotometric profiles, KO and UBKO may be considered supporting ingredients in sunscreen formulations.

The photoprotective potential of UBKO could result from complementary mechanisms that combine chemical UV absorption with advanced biological cytoprotection. Karanja oil is rich in furanoflavonoids, mainly karanjin and pongamol. These natural phytocompounds act as UVA and UVB filters, absorbing radiation directly in the 290–400 nm range, which explains the calculated high baseline SPF of 30. UV radiation generates reactive oxygen species (ROS), which cause lipid peroxidation and cellular DNA damage. Usnea barbata, with its high content of usnic acid and polyphenols, functions as a secondary antioxidant [76,77,78]. It neutralizes UV-induced ROS, prevents secondary oxidative stress, and inhibits inflammatory cascades such as cyclooxygenase-2 and pro-inflammatory cytokines, serving as a biologically active cellular protector (bio-photoprotector) [76,79]. Although the in vitro spectrophotometric Mansur method is widely accepted and highly reproducible for evaluating Sun Protection Factor (SPF), it primarily measures a formulation’s passive UV-filtering capacity [80,81]. In our study, the SPF value of 30 achieved by UBKO can be attributed to a complementary contribution of the lichen metabolites. Karanja oil inherently provides a robust structural shield against both UVB and UVA radiation due to its high concentration of furanoflavonoids, such as karanjin and pongamol, which contain chromophores that dissipate absorbed photons [4]. At the same time, adding Usnea barbata metabolites during the extraction process provides an important secondary layer of biological photoprotection. Standardized lichen metabolites, especially (+)-usnic acid and related polyphenols, have strong radical scavenging abilities. When exposed to ultraviolet radiation, these compounds reduce UV-induced cellular damage by neutralizing reactive oxygen species (ROS) and by lowering pro-inflammatory pathways, such as TNF-alpha and NF-kB expression, which cause solar erythema [70]. So, the UBKO-based further formulations not only act as a passive ultraviolet barrier but also as a bioactive, cytoprotective system that can reduce both optical penetration and oxidative stress at the biomolecular level [19,82]. Additionally, the passive screening results obtained with the Mansur method (SPF = 30) should be correlated with UBKO’s free radical-scavenging ability, as assessed via DPPH and ABTS methods, because UV radiation triggers intracellular oxidative stress, initiating a cascade of singlet oxygen and hydroxyl radicals that can overwhelm the skin’s natural defenses. However, our findings represent baseline in vitro UV-absorbing trends, and further in vivo verification is needed to determine their actual photoprotective relevance.

Beyond bioactivity, an important aspect of this lichen extract in KO is its physicochemical compatibility. Usnic acid is lipophilic; it is nearly insoluble in water but dissolves easily in oils and organic solvents [53]. Karanja oil, rich in oleic acid, naturally solubilizes lipophilic polyphenols. Hence, KO could serve as an ideal carrier for usnic acid, potentially enhancing its skin bioavailability by promoting penetration, as demonstrated with U. barbata extract in Canola oil [83]. Lipid-based systems address the instability and poor solubility of lipophilic actives. Nanostructured lipid carriers co-encapsulating flavonoids can achieve complementary antimicrobial effects against multidrug-resistant pathogens.

Major concerns center on usnic acid as a well-known contact allergen that can trigger type IV hypersensitivity, mainly documented through patch tests in patients exposed to natural cosmetics or occupational lichen contact [84]. Its growing presence in botanical personal care products warrants greater clinical caution [85]. Therefore, topical formulations containing usnic acid should undergo additional testing in patients with dermatitis on the axillae, face, or sun-exposed areas using botanical products. However, lichen additives are increasingly causing allergic contact dermatitis, with usnic acid and oak moss absolute (Evernia prunastri) among the most common culprits [85,86,87]. Usnic acid functions as a classic low-molecular-weight hapten. Skin sensitization occurs through covalent binding of these chemicals to proteins, a process known as haptenation [88]. This involves two phases: (1) first, sensitization, in which the hapten penetrates the stratum corneum, interacts with keratinocytes, and triggers the release of proinflammatory cytokines; (2) second, elicitation, in which re-exposure activates effector T cells. The hapten–carrier complex becomes a new self-antigen, stimulating both B and T cells when presented by HLA molecules, leading to a delayed-type (type IV) hypersensitivity response that generally appears 48–72 h after re-exposure [89,90]. However, Karanja oil’s emollient fatty acids can help counteract the potential drying or irritating effects of usnic acid at higher concentrations, improving tolerability—especially in sensitive-skin formulations. A rigorous toxicological characterization, including in vivo skin-irritation tests, will be conducted in our future research, aiming to incorporate UBKO into various topical formulations.

5. Conclusions

Extracting Usnea barbata metabolites into Pongamia pinnata (Karanja) seed oil significantly enhances its properties. The present study showed noticeable improvements in phenolic-equivalent content and antimicrobial activity (especially against S. aureus and C. albicans), along with a slight increase in SPF and oxidative stability. Our results suggest that UBKO could serve as a multifunctional ingredient in sunscreen formulations.

Complex biological tests are needed to confirm UBKO’s photoprotective potential and other skincare benefits. Additional experiments should investigate the effects of usnic acid and karanjin on S. aureus and other bacterial and fungal pathogens that cause skin infections, to better understand their interactions. Further research should also assess whether Karanja oil improves usnic acid delivery compared to water-based systems and include ex vivo experiments using inflammatory skin models, comparing formulations with both ingredients to each ingredient alone.

Moreover, advanced dermatological compatibility testing, toxicological assessments, acute dermal irritation, skin sensitization, phototoxicity, and in vitro cytotoxicity are required for the development and optimization of cosmetic formulations.