Photochemical UV-induced modification of amber oil, enhancing antimicrobial, anti-inflammatory, and wound healing activities with molecular docking insights

Yahya, R., Al-Rajhi, A. M. H., Alghonaim, M. I., Daws, D., Kashmery, H. A., Alruhaili, M. H., Gattan, H. S., and Selim, S. (2026). "Photochemical UV-induced modification of amber oil, enhancing antimicrobial, anti-inflammatory, and wound healing activities with molecular docking insights," BioResources 21(4), 10425–10448.

Abstract

This study investigated the photochemical transformation of amber oil under UV irradiation and its impact on biological activities. Gas chromatography–mass spectrometry analysis showed that untreated amber oil was rich in long-chain hydrocarbons and fatty acids, predominantly cis-vaccenic acid (36.53%), whereas UV irradiation produced oxygenated compounds, mainly 2-(2-hydroxypropoxy)-1-propanol (13.02%), 1,1′-oxybis-2-propanol (6.93%), and diethyl phthalate (6.73%). UV-treated oil exhibited enhanced antimicrobial activity, particularly against Bacillus subtilis (22 ± 1.0 mm; MIC 15.62 µg/mL) and Candida albicans (24 ± 0.5 mm; MIC 15.62 µg/mL). Strong antibiofilm activity was recorded, exceeding 90% inhibition at 75% MBC against tested bacteria, including Staphylococcus aureus and Escherichia coli. Anti-inflammatory evaluation showed effective inhibition of protein denaturation (IC₅₀ = 2.60 ± 0.11 µg/g). Wound healing assays showed a high closure (90.9%) for UV-treated oil, compared to untreated oil (61.1%). Molecular docking revealed that cis-vaccenic acid exhibited stronger binding affinity toward the target proteins of Aspergillus terreus (4KWD) and B. subtilis (3OIF) than 2-(2-hydroxypropoxy)-1-propanol, with docking scores of −6.64 and −7.12 kcal/mol, respectively. These findings demonstrate that UV-induced photochemical modification is an effective approach to enhance the biological properties of amber oil, providing a basis for its further investigation in natural bioactive product applications.

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Photochemical UV-Induced Modification of Amber Oil, Enhancing Antimicrobial, Anti-Inflammatory, and Wound Healing Activities with Molecular Docking Insights

Reham Yahya,^a,b Aisha M. H. Al-Rajhi,^cMohammed Ibrahim Alghonaim,^d,* Dalia Daws,^eHeba A. Kashmery,^f Mohammed H. Alruhaili,^g,i Hattan S. Gattan,^h,i and Samy Selim ^j,*

DOI: 10.15376/biores.21.4.10425-10448

Keywords: Amber oil; UV irradiation; Antibiofilm activity; Scratch assay; Protein denaturation

Contact information: a: Basic Sciences Department, College of Science and Health Professions, King Saud Bin Abdulaziz University for Health Sciences, Riyadh, 11671, Saudi Arabia; b: King Abduallah International Medical Research Center, P.O. Box 3661, Riyadh, 11481, Saudi Arabia; c: Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, 11671 Riyadh, Saudi Arabia; d: Department of Biology, College of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU), P.O. Box 90950 Riyadh 11623, Saudi Arabia; e: Pharmacy Department, Jazan University Hospital, Jazan University, Jazan, Saudi Arabia; f: Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia; g: Department of Clinical Microbiology and Immunology, Faculty of Medicine, King Abdulaziz University, Jeddah 21589, Saudi Arabia; h: Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, King Abdulaziz University, Jeddah 21589, Saudi Arabia; i: Special Infectious Agents Unit, King Fahad Medical Research Center, King AbdulAziz University, Jeddah, Saudi Arabia; j: Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, Jouf University, Sakaka, Saudi Arabia;

* Corresponding author: [email protected] (M.I.A.), [email protected] (S.S.)

INTRODUCTION

Although plant extracts and essential oils possess inherent biological activity, recent technological advancements have significantly improved their efficacy. Studies have demonstrated that ultraviolet (UV) irradiation can induce structural modifications in oil constituents such as sage oil (Alsalamah et al. 2025a), Aloe vera oil (Alsalamah et al. 2025b), and pumpkin seed oil (Alsalamah et al. 2025c), leading to the formation of more potent bioactive compounds. Similarly, ozone (O₃) treatment of black seed oil (Al-Rajhi et al. 2024), propolis (Al-Rajhi et al. 2025a), and peanut oil (Al-Rajhi et al. 2025b) has proven effective in enhancing antimicrobial activity by oxidizing certain components and converting them into more reactive forms. Furthermore, infrared radiation or ultrasonic-assisted extraction techniques can increase extraction efficiency by disrupting plant cell walls, thereby releasing a greater quantity of active phytochemicals.

Recent research has demonstrated that amber oil from plant resin has important biological qualities, such as antioxidant, antibacterial, and neuro-calming effects, in addition to its fragrant value. The presence of bioactive substances including ambroxide, ambrox, squalene, and other terpenoid components is primarily responsible for these benefits and for the plant’s possible use in medicine and cosmetics. Additionally, a number of studies indicate that amber oil’s hydrating and skin-elasticity-boosting qualities make it suitable for use in skincare formulas. In folk medicine, it has also long been used to treat sleep issues, stress, and headaches. Amber oil derivatives may now be obtained using safer and more environmentally friendly ways due to the development of current extraction technology, which opens up new possibilities for both industrial and medicinal use (Levchyk et al. 2017; Al-Tamimi et al. 2020). Several cutting-edge extraction and purification processes have been used in several research investigations aimed at increasing the medicinal value of amber oil in order to enhance the levels bioactive substances such as terpenoids and succinic acid, hence increasing antioxidant effectiveness (Sogo et al. 2021).

A computer method called molecular docking is used to estimate the strength and type of a ligand-target protein interaction by predicting the ligand’s preferred orientation when bound to the target protein. This approach uses algorithms that assess binding affinity by considering the ligand-receptor spatial complementarity and energy minimization (Al-Rajhi et al. 2022). Researchers can determine which ligand–protein complexes are the most stable and examine important interactions including electrostatic forces, hydrophobic contacts, and hydrogen bonds by modeling the docking process (Qanash et al. 2022; Binsaleh et al. 2025). The significance of molecular docking is rooted in its capacity to offer essential insights into the molecular mechanisms that drive biological activity, thereby informing the design and refinement of bioactive compounds. It acts as a cost-efficient and time-saving substitute for experimental screening, facilitating the prediction of potential drug targets, clarification of binding modes, and comprehension of structure–activity relationships (Alsalamah et al. 2023).

The present investigation aimed to examine the effect of UV irradiation on the chemical composition and biological activities of amber oil, including its antimicrobial, antibiofilm, anti-inflammatory, and wound-healing properties, supported by molecular docking studies. The findings are expected to provide valuable insights into the use of UV-induced photochemical transformation as a simple and effective approach for enhancing the therapeutic potential of amber oil, thereby supporting its future development as a natural bioactive ingredient for pharmaceutical and biomedical applications.

EXPERIMENTAL

Employed Chemicals and UV Irradiation of Amber Oil

Commercial amber oil was sourced from Alhedaia Company (Egypt), a manufacturer of natural oils and herbal cosmetic ingredients. The oil was divided into two equal portions (1 L each), one of which was exposed to ultraviolet-B (UV-B) light for 30 min, while the other portion was not exposed to UV-B. Irradiation was carried out using a SUN V-17/F UV lamp operating at 235 V, 50 Hz, and 0.16 A, with a peak emission at 280 nm. All other analytical-grade chemicals and reagents employed in this study were obtained from Sigma (Egypt).

Chemical Characterization of UV-Treated and Untreated Amber Oil by GC–MS

The chemical ingredients of UV-irradiated and non-irradiated amber oil was examined employing an Thermo Scientific ISQ 7000 GC–MS system. Each oil sample was diluted to approximately 1% (v/v) in n-hexane prior to injection. A 1.0 µL aliquot of the prepared solution was introduced into the injector operating in split mode (split ratio 1:50). Helium served as the carrier gas at a steady flow rate of 1.0 mL/min. The oven temperature was initially set at 45 °C for 1 min, then raised at a rate of 3 °C/min to 200 °C (held for 3 min), followed by a further increase to 280 °C at the same rate and maintained for 10 min. The injector and ion source temperatures were maintained at 250 °C, while the transfer line was set to 240 °C. The mass spectrometer operated in electron ionization (EI) mode, scanning over an m/z range of 50 to 1000 with a scan rate of 0.2 s per spectrum. Compound identification was achieved by comparing the obtained mass spectra and retention times with those in reference spectral libraries (e.g., NIST) and literature data. The relative abundance of each compound was calculated from its peak area and expressed as a percentage of the total ion current (Al-Rajhi and Abdelghany 2023).

Protein Denaturation Inhibition Assay Using Bovine Serum Albumin

The anti-denaturation potential of UV-treated and untreated amber oil was determined using Bovine Serum Albumin (BSA) as the standard protein model, following a previously reported procedure with slight modifications. A series of test concentrations (1.56 to 200 µg/mL) of each oil sample were prepared. To each reaction tube, 50 µL of the test sample was combined with 450 µL of 1% (w/v) BSA solution prepared in distilled water. The pH of the mixture was adjusted to 6.3 using 1 N HCl. The reaction mixtures were allowed to equilibrate at room temperature for 20 minutes, followed by incubation in a water bath at 55 °C for 30 min to induce thermal denaturation. After cooling to ambient temperature, the absorbance (A) of each sample (test) was measured at 670 nm using a Biosystem 310 Plus spectrophotometer (Al-Rajhi et al. 2025a). Diclofenac sodium served as the standard reference anti-inflammatory drug. The extent of protein denaturation inhibition was determined as follows (Eq. 1):

Wound-closure Assay and Analysis

Cells (HFB4 human normal skin fibroblast cell line) were grown to confluent monolayers in 6-well plates under standard conditions (37 °C, 5% CO₂) in appropriate complete medium. A sterile 200-µL pipette tip was used to create a straight scratch (“wound”) across each well. Detached cells were removed by gently washing with PBS and fresh serum-reduced medium was added. Wells were treated with: (1) Control (vehicle only), (2) UV-untreated amber oil, and (3) UV-treated amber oil. Treatments were applied immediately after scratch (time 0 h). All treatments were performed in biological replicates (n = 6). Images of the same wound area were captured at 0 h and 48 h using an inverted phase-contrast microscope with a calibrated objective. Wound width and wound area were measured from images using ImageJ (Fiji) “Measure” tools after thresholding and outlining the wound region. All distances are reported in micrometres (µm) and areas in µm² (ImageJ calibration performed with a stage micrometer) (Bakri et al. 2024). Remaining width at 48 h (Remaining width, RM, µm) = measured mean wound width at 48 h (µm).

Agar Well Diffusion Assay for Antimicrobial and Antifungal Activity with MIC, MBC, and MFC Determination

The antimicrobial efficacy of UV-treated and untreated amber oil was assessed using the agar well diffusion method against selected bacterial and fungal strains (Al-Rajhi and Abdelghany 2023; Bazaid et al. 2025). The strains included Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538), Klebsiella pneumoniae (ATCC 13883), Salmonella typhi (ATCC 6539), Candida albicans (ATCC 10221), and Aspergillus terreus (AUMC 15762). Bacterial inocula were standardized to approximately 1 × 10⁸ CFU/mL (equivalent to 0.5 McFarland standard), and fungal suspensions were adjusted (1 × 10⁶ spores/mL). Mueller–Hinton agar (MHA) was used for bacterial assays and Sabouraud dextrose agar (SDA) for fungal assesses. The sterile media plates were inoculated uniformly by dispersion 100 µL of each microbial suspension over the surface using a sterile cotton swab. Wells of 6 mm diameter were aseptically bored into the agar employing a sterile cork borer. Each well was filled with 50 µL of UV-treated amber oil, untreated amber oil, and the respective standard antimicrobial (Ciprofloxacin,10 µg/mL) or antifungal (fluconazole, 25 µg/mL) drugs. Dimethyl sulfoxide (DMSO) was used to dissolve the samples and served as the negative control at 1 %. Plates were left at room temperature for 30 min to allow proper diffusion of the samples into the agar, then incubated under appropriate conditions: 37 °C for 24 h for bacteria, 28 °C for 48 to 72 h for fungi. After incubation, the clear inhibition zones around the wells were measured in millimeters using a digital caliper.

The minimum inhibitory concentration (MIC) of UV-treated and untreated amber oil was assayed by the broth microdilution method following CLSI guidelines. Serial two-fold dilutions (15.625 to 500 µg/mL) of each sample were prepared in 96-well microplates using Mueller–Hinton broth for bacteria and Sabouraud dextrose broth for fungi. Each well received 100 µL of test dilution and 100 µL of standardized inoculum (1×10⁶ CFU/mL for bacteria; 1×10⁵ spores/mL for fungi). Plates were incubated at 37 °C for 24 h (bacteria) and 28 °C for 48 to 72 h (fungi). The MIC was recorded as the lowest concentration showing no visible growth (Qanash et al. 2025). For MBC (Minimum Bactericidal Concentration) and MFC (Minimum Fungicidal Concentration), 10 µL from clear wells were cultured on fresh agar plates; the lowest concentration producing no colony growth was considered the MBC or MFC.

Microplate-based Biofilm Inhibition Assay

The antibiofilm activity of UV-treated and untreated amber oil was evaluated using the microtiter plate method with minor modifications. Overnight cultures of the test bacteria were standardized to approximately 1×10⁶ CFU/mL in freshly prepared tryptic soy–yeast extract (TSY) broth. Aliquots of 150 µL of the inoculated medium were transferred into sterile, flat-bottom 96-well polystyrene microplates and immediately mixed with an equal volume (150 µL) of the oil solution prepared at 25%, 50%, and 75% of its previously established MBC, resulting in a final volume of 300 µL per well. For each experiment, uninoculated medium served as a blank control, inoculated medium without oil as the growth control, and inoculated medium containing the highest solvent concentration as the solvent control. The plates were incubated statically at 37 °C for 48 h to promote biofilm formation. Following incubation, the supernatant containing planktonic cells was carefully aspirated, and the wells were gently washed three times with sterile distilled water to remove non-adherent cells. The plates were then air-dried at room temperature for 30 min. Subsequently, biofilms were stained with 0.1% (w/v) crystal violet solution for 15 min, and excess dye was removed by rinsing the wells three times with sterile distilled water. The bound stain was then solubilized with 250 µL of 95% ethanol per well and left to stand for 15 min. Absorbance (A) values were measured at 570 nm using a microplate reader (Selim et al. 2024). The degree of biofilm formation inhibition was evaluated using Eq. 4:

Docking Investigation

The docking study was conducted using MOE 2019.01 (Chemical Computing Group, Montreal, Canada). Crystal structures of A. terreus protein (PDB ID: 4KWD) and B. subtilis protein (PDB ID: 3OIF) were retrieved from the Protein Data Bank. The proteins were prepared by removing water molecules, addition of hydrogen atoms, assignment of partial charges using the AMBER99 force field, and energy minimization until an RMSD gradient of 0.05 kcal/mol/Å² was achieved (Al Abboud et al. 2024).

Ligand preparation

The 3D structures of 2-(2-hydroxypropoxy)-1-propanol and cis-Vaccenic acid were constructed using the MOE builder module. Geometry optimization was performed using the MMFF94x force field with a convergence criterion of 0.0001 kcal/mol. Partial charges were calculated using the AM1-BCC method.

Docking protocol

Docking was performed using the Triangle Matcher placement method and London dG scoring function for initial placement, followed by GBVI/WSA dG refinement.

For each ligand-protein complex, 30 different poses were generated and ranked based on their docking scores (S). The top 5 poses for each ligand-protein pair were selected for further analysis.

Interaction analysis

Protein-ligand interactions were analyzed using MOE’s Ligand Interactions module. Hydrogen bonds, hydrophobic interactions, and other non-covalent interactions were identified and characterized by distance and energy values. Interaction diagrams were generated to visualize the binding modes.

Statistical Analysis

Each experimental measurement was achieved in triplicate, and the obtained data were offered as mean values convoyed by their standard deviations (mean ± SD). Statistical assessment of differences among treatments was carried out through one-way ANOVA, and pairwise comparisons were assessed using Tukey’s multiple range test. All analyses were achieved with GraphPad Prism software (version 9.0), and significance was accepted at P < 0.05.

RESULTS AND DISCUSSION

Phytochemical of Treated and Untreated Amber Oil by UV Irradiation

The GC–MS chromatogram of the amber oil before UV-induced photochemical treatment (Fig. 1, Table 1) displays a complex array of long-chain hydrocarbons, fatty acids, alcohols, aldehydes, terpenoids, and sterols, highlighting the varied composition of the native oil. The primary identified compounds comprised cis-vaccenic acid (36.5%), hexadecanoic acid (4.69%), α-Sitosterol (4.60%), along with several oxygenated hydrocarbons such as 9-eicosyne (0.97%) and 1-heptatriacotanol (0.31%). The profile also indicated the presence of phenolic and aromatic compounds, including isochiapin B (0.46%), sesamin (0.27%), and flavone glucosides, which suggest the antioxidant potential inherent in the untreated sample. In contrast, the UV-treated amber oil (Fig. 2; Table 2) demonstrated a significant alteration in chemical composition, reflecting photochemical transformation and oxidation of various long-chain aliphatic molecules into smaller, more oxygenated derivatives. The treated sample was characterized by low-molecular-weight oxygenated compounds, such as 1-propanol derivatives (13.02%), 2-propanol analogs (6.93%), and 2-keto-D-gluconic acid (3.22%), which were not present in the untreated oil. Furthermore, new esters and alcohols, including 1,2-benzenedicarboxylic acid, diethyl ester (6.73%), tetraethylene glycol monomethyl ether (0.97%), and patchouli alcohol (0.25%), emerged following UV exposure, indicating oxidative cleavage and esterification reactions. Notably, the levels of fatty acids and sterols diminished in the treated oil, where n-hexadecanoic acid (0.74%) and trans-13-octadecenoic acid (3.03%) were found in reduced quantities compared to the untreated oil. However, UV exposure facilitated the formation of new bioactive sterol analogs such as stigmasterol (0.68%) and 3-docosenamide (12.5%), both linked to anti-inflammatory and antioxidant properties. In summary, UV irradiation simplified the hydrocarbon matrix while increasing the prevalence of oxygenated and polar constituents. This transformation indicates that UV treatment facilitates photo-oxidation, resulting in the creation of new esters, alcohols, and acids that may exhibit enhanced biological activity. The presence of these newly functionalized molecules could account for the superior biological activities such as antimicrobial and anti-inflammatory characteristics noted in the UV-treated amber oil in comparison to the untreated sample. Under the applied UV irradiation conditions, photochemical oxidation promoted the conversion of long-chain hydrocarbons and fatty acids into smaller oxygen-containing molecules, including alcohols, esters, and organic acids. This compositional remodeling was accompanied by a decrease in hydrophobic constituents and an increase in polar oxygenated compounds, which are generally expected to exhibit greater chemical reactivity and improved interactions with biological targets. Such changes may enhance membrane permeability, facilitate interactions with microbial cell components, and improve binding to inflammation-related proteins. Accordingly, the chemical modifications induced by UV irradiation corresponded with the enhanced biological activities observed experimentally, including increased antimicrobial and antibiofilm activities, improved inhibition of protein denaturation, and accelerated wound closure. Although the contribution of individual compounds was not experimentally verified, the collective enrichment of oxygen-containing constituents provides a plausible explanation for the superior biological performance of UV-treated amber oil. These findings are consistent with previous reports demonstrating that controlled UV irradiation can beneficially modify the composition and bioactivity of natural oils while excessive irradiation may promote degradation of key constituents (Shahzaidi et al. 2025; Arcos-Limiñana et al. 2025; Gulcin 2025).

Fig. 1. GC-MS chromatogram of untreated amber oil by UV irradiation

Fig. 2. GC-MS chromatogram of treated amber oil by UV irradiation

Table 1. GC-MS Analysis Constituents of Untreated Amber Oil by UV Rays

Table 2. GC-MS of Analysis of Constituents Treated Amber Oil by UV Rays

Anti-inflammatory Activity of Treated and Untreated Amber Oil by UV Irradiation

The anti-inflammatory capabilities of both UV-treated and untreated amber oil were evaluated through the protein denaturation inhibition assess, with diclofenac sodium utilized as the reference (Table 3). At the lowest dose (1.56 µg/g), diclofenac sodium demonstrated a 45.6% inhibition rate, whereas the treated and untreated amber oils exhibited slightly lower inhibition rates of 43.9% and 41.7%, respectively. This pattern was consistent across all concentrations, suggesting that both types of amber oil possess anti-inflammatory properties, albeit with efficacy slightly inferior to that of the diclofenac sodium. Statistical analysis validated that the differences between diclofenac sodium and both oil samples were significant (P < 0.05) at most doses. Notably, the UV-treated oil consistently showed marginally higher %inhibition compared to the untreated oil, indicating that UV irradiation may have enhanced the anti-inflammatory potential of the oil. This enhancement could be due to photochemical changes of the oil induced by UV exposure. The calculated IC₅₀ values further corroborate these observations: diclofenac sodium had the lowest IC₅₀ (1.75 ± 0.08 µg/g), followed by untreated amber oil (2.14 ± 0.10 µg/g) and UV-treated oil (2.60 ± 0.11 µg/g). Although both oils presented slightly higher IC₅₀ values than the reference drug, indicating reduced potency, their inhibitory effects remained significant and statistically comparable. The enhanced performance of the UV-treated oil may be associated with the structural oxidation of unsaturated fatty acids and terpenoids, leading to the formation of compounds with a greater affinity for protein-binding sites and improved stability under physiological conditions. The inhibition of protein denaturation by both oils suggests their capability to minimize the denaturation of biological proteins, a common mechanism linked with inflammatory responses. The strong anti-inflammatory activity of the amber oil is attributed to its ability to inhibit the NF-κB p65 signaling pathway (Tian et al. 2021). This effect is further linked to a decrease in intracellular reactive oxygen species (ROS) production. Besides, a significant suppression of heat- or chemical-induced protein denaturation, a crucial indicator of inflammation, has been shown in vitro studies assessing the anti-inflammatory properties of natural oil and different vegetable oils using protein denaturation assays. Significant albumin and egg protein structural stabilization was demonstrated by amber oil, as well as oils such as olive, coconut, and sesame, suggesting membrane-protective action. Fatty acids, terpenoids, and phenolics all played a part in this suppression. Previous studies have demonstrated that certain essential oils possess promising anti-inflammatory properties, highlighting their potential as natural sources of anti-inflammatory agents (Lima et al. 2024; Batovska et al. 2025). Furthermore, the effects of UV exposure on oils and their anti-inflammatory action show that, depending on the exposure settings, UV treatment can either increase or decrease its bioactivity. It has been demonstrated that short-term, controlled UV irradiation increases anti-inflammatory potential by producing novel oxidized compounds that enhance protein stability. But excessive or protracted UV radiation reduced the prevention of protein denaturation by degrading phenolic chemicals and important fatty acids. After irradiation, structural alterations in lipid and antioxidant components were verified by spectroscopic studies (Chittasupho et al. 2022; Tsiapali et al. 2025).

Table 3. Anti-inflammatory Activity of UV-treated and Untreated Amber Oil Compared with Diclofenac Sodium based on Inhibition of Protein Denaturation

Wound Healing Activity of Treated and Untreated Amber Oil by UV Irradiation

The wound healing efficacy of both UV-treated and untreated amber oil was evaluated by tracking the reduction in wound width and area over a period of 48 h (Table 4 and Fig. 3). The findings indicated a notable enhancement in wound closure for both oil-treated groups in comparison to the control group (P < 0.05), with the UV-treated amber oil demonstrating the most significant effect. After 48 h, the control group showed a mean wound area of 540,000 µm², which corresponds to a closure percentage of 56.8%. The untreated amber oil group displayed a marginally higher closure of 61.1%, with a mean residual area of 676,000 µm² and a wound margin (RM) of 11.1 µm. In contrast, the UV-treated amber oil group exhibited a significantly greater reduction in wound area, achieving a mean residual area of 114,000 µm², smaller mean wound width (106 µm), and a wound closure of 90.9%, which was statistically significant when compared to both the control and untreated groups (P < 0.05), indicating accelerated tissue regeneration and epithelialization relative to the untreated oil and control groups.

Table 4. Wound Healing Activity of UV-treated (T), Untreated (UT) Amber Oil and without T or UT (C)

Fig. 3. Wound healing activity of UV-treated (C) and untreated (B) amber oil compared control cells (A)

The improved healing efficiency of the UV-treated amber oil may be ascribed to photochemical changes induced by UV exposure, resulting in the generation of oxygenated derivatives such as esters, alcohols, and phenolic compounds recognized in the GC–MS profile (Table 2). These compounds are recognized for their ability to enhance collagen synthesis, diminish oxidative stress, and expedite keratinocyte migration—all vital processes in wound closure. In summary, the results suggest that UV-treated amber oil possesses significant wound healing properties, markedly superior to those of untreated oil or the control. This improvement highlights the potential of UV modification as an environmental-friendly and effective approach to enhance therapeutic efficacy. According to in vitro research on the wound-healing properties of UV-treated and untreated natural oils, both types stimulate collagen formation, cell migration, and proliferation in fibroblast and keratinocyte cells. The natural terpenoids and succinic acid found in untreated oil promote wound healing by lowering inflammation and boosting antioxidant protection. Because UV-treated oil produces new oxygenated molecules that promote the development of growth factors and extracellular matrix, it exhibits enhanced cellular regeneration when exposed under regulated conditions. On the other hand, too much UV light can break down active ingredients, decreasing their effectiveness. All things considered, mildly UV-treated oil exhibits better in vitro wound healing capability than untreated oil (Trigo et al. 2024; Al-Rajhi et al. 2025b).

Antimicrobial Activity of Treated and Untreated Amber Oil by UV Irradiation

Table 5 and Fig. 4 illustrate the antimicrobial efficacy of both UV-treated and untreated amber oil in comparison to standard antimicrobial agents against various pathogenic bacteria and fungi. From the outcomes, the UV-treated amber oil demonstrated greater antimicrobial effectiveness than the untreated variant. This was reflected in the larger inhibition zone diameters and reduced MIC and MBC or MFC values. Among the bacterial strains, B. subtilis exhibited the highest sensitivity to both the treated and untreated amber oil, with inhibition zones measuring 22 ± 1.0 mm and 20 ± 0.4 mm, respectively. The MIC and MBC values of the treated oil (15.6 µg/mL) were reduced to half of those of the untreated sample (31.2 µg/mL), signifying a two-fold enhancement in potency following UV exposure. In a similar manner, K. pneumoniae and S. typhi exhibited increased sensitivity to the treated oil, as evidenced by larger inhibition zones (15 ± 0.8 mm and 16 ± 0.5 mm, respectively) and lower MIC/MBC values (31.25 µg/mL) in comparison to the untreated variant (62.5 µg/mL). S. aureus demonstrated moderate sensitivity, with only minimal differences observed between the UV-treated amber oil and the untreated amber oil (before UV irradiation), which produced inhibition zones of 15 ± 0.2 mm and 14 ± 0.6 mm, respectively. These findings indicate that UV irradiation had only a limited effect on the antibacterial activity against this organism. Concerning fungal strains, both C. albicans and A. terreus were inhibited by amber oil, with significantly enhanced effects noted post-UV treatment. The inhibition zone for C. albicans increased from 21 ± 0.5 mm to 24 ± 0.5 mm, while the MIC decreased from 31.2 µg/mL to 15.6 µg/mL.

The most significant improvement was observed for A. terreus, where the inhibition zone rose from 17 ± 0.2 mm (untreated) to 24 ± 1.0 mm (treated), along with a marked reduction in MIC and MFC values (from 125 µg/mL to 62.5 µg/mL and from 62.5 µg/mL to 31.25 µg/mL, respectively). DMSO (1%, v/v) showed no detectable antimicrobial activity against any of the tested microorganisms under the experimental conditions (Fig. 4). By producing oxygenated and peroxidized compounds that more effectively rupture microbial cell membranes and result in lower MIC values, controlled UV treatment can increase this activity. Long-term or intense UV exposure, however, may weaken important bioactivities and reduce their efficacy. In comparison to their untreated equivalents, mildly UV-treated natural oils appear to have better antimicrobial and MIC efficacy (Kebede and Shibeshi 2022; Shehabeldine et al. 2023; Alsakhawy et al. 2024; Al-Rajhi et al. 2024).

The antibiofilm activity of both UV-treated and untreated amber oil against the examined bacteria at varying doses of 25%, 50%, and 75% of their respective MBC values was recorded (Fig. 5). In general, both UV-treated and untreated amber oils displayed a dose-dependent inhibition of biofilm formation across all tested bacteria. Nevertheless, the UV-treated oil exhibited a significantly greater antibiofilm efficacy at all tested concentrations when compared to the untreated oil, suggesting that UV exposure has enhanced the bioactive properties of amber oil. At 25% of MBC, the biofilm inhibition varied from 37.8% to 65.8% for K. pneumoniae, and from 43.4% to 72.5% for S. typhi when using untreated and UV-treated amber oil, respectively. The treated oil also demonstrated substantial inhibition against S. aureus and B. subtilis, achieving 74.7% and 74.0%, respectively, in contrast to 62.7% and 47.5% for the untreated oil. Increasing the dose to 50% of MBC significantly improved biofilm inhibition, achieving 89.0% for S. aureus, 83.8% for S. typhi, 80.7% for K. pneumoniae, and 84.4% for B. subtilis in the treated samples. The untreated samples displayed considerably lower values, ranging from 54.5% to 77.9%. At the maximum tested concentration of 75% of MBC, UV-treated amber oil demonstrated over 90% inhibition across all bacterial species, with the highest inhibition noted for B. subtilis (94.5%) and S. aureus (94.1%).

This nearly complete inhibition of biofilm formation underscores the strong antibiofilm potential of the UV-modified oil. These observations align with Fig. 6, which visually depicts the reduction in biofilm density in microplates subjected to increasing concentrations of amber oil. The enhancement of antibiofilm activity following UV treatment may be due to photo-induced chemical alterations in the components of amber oil, resulting in the creation of new reactive or antimicrobial compounds that hinder bacterial adherence and the production of extracellular polymeric substances (EPS). The application of UV treatment markedly improved the antibacterial and antibiofilm characteristics of amber oil, in addition to significant antifungal properties. This enhancement is probably attributable to UV-induced alterations in the chemical composition of the oil, which resulted in the generation or activation of more effective bioactive compounds. Therefore, UV-modified amber oil presents considerable promise as a natural broad-spectrum antimicrobial and antibiofilm agent for use in biomedical and industrial fields. UV radiation can have a substantial impact on the antimicrobial effectiveness of natural oils, according to in vitro research examining the antibiofilm and MIC activities of both UV-treated and untreated natural oils.

Untreated oils exhibit natural antibiofilm qualities by interfering with the production of EPS and bacterial adherence. By generating oxygenated terpenoid and peroxide derivatives that penetrate and degrade established biofilms, controlled UV treatment amplifies these effects and lowers MIC and biofilm inhibition concentrations. Excessive UV exposure, however, might weaken essential chemicals and decrease their effectiveness. When compared to untreated samples, appropriately UV-treated natural oils exhibit superior antibiofilm and antibacterial effectiveness (Iseppi et al. 2020; Diouchi et al. 2024).