Antimicrobial and Antioxidant Properties of Sambucus nigra L. (Elderflower) Oil: A Molecular Docking and Biochemical Study

Abstract

The present study investigates the antimicrobial and antioxidant potential of an essential oil extracted from Sambucus nigra L. flowers. Using hydrodistillation, the volatile compounds were profiled through GC–MS analysis for the fatty acid profile and volatile compounds. The fatty acid profile demonstrated a balanced composition of saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids, with oleic, palmitic, and linolenic acids as key contributors. The volatile profile revealed the dominance of nonanal, cis-rose oxide, trans-rose oxide, and 2-Pentadecanone, 6,10,14-trimethyl-. Antioxidant activity was assessed using the 1,1-Diphenyl-2-Picrylhydrazyl radical scavenging assay, showing significant inhibition, with an IC₅₀ value of 2.52 mg/mL. Antimicrobial efficacy was determined against Gram-positive, Gram-negative, and fungal strains, highlighting moderate inhibitory activity for Streptococcus pyogenes, Staphylococcus aureus, and Candida albicans. The S. nigra essential oil exhibited more activity against fungal strains, especially C. albicans, compared to the bacterial strains, which might be attributed to differences in the composition and permeability of the cell wall between fungi and bacteria. Among the bacteria, E. coli was the most susceptible, while P. aeruginosa showed moderate resistance, in agreement with its known stronger membrane structure and efflux mechanisms. Molecular docking analysis was conducted to evaluate the potential inhibitory effects of the oil on microbial proteins to corroborate the observed in vitro outcome. The results indicated that nonanal, cis-rose oxide, trans-rose oxide, and 2-pentadecanone, 6,10,14-trimethyl- displayed interesting hydrophilic and hydrophobic binding interactions with the putative microbial proteins. These findings elucidate the bioactive role of Sambucus nigra essential oils, suggesting their potential as therapeutic agents in managing oxidative stress and microbial infections.

1. Introduction

The use of medicinal plants as remedies for various pathologies due to their bioactive compounds has been reported since ancient times. Although modern medicine has long disproved their biological potential, currently, research is based on the identification of some natural bioactive compounds from medicinal plants that, on the one hand, have a beneficial effect on the body’s energy and vitality and on the other hand, replace the synthetic products used for the treatment of diseases [1,2,3,4,5].

Sambucus L. is a genus of the family Adoxaceaein, consisting of more than 20 flowering plants that grow from the temperate to subtropical regions of the world [6,7]. S. nigra, or black elderberry, is a deciduous shrub native to Europe, North Africa, and parts of Asia. Thriving in temperate regions with moist soils, it has been valued for centuries for its medicinal and culinary uses. Over time, it spread to other parts of the world, including North America, becoming both naturalised and integral to herbal medicine and folklore. Among the Sambucus species, the most cultivated and studied are Sambucus nigra L. (native to Europe and western Asia) and S. nigra ssp. canadensis (native to eastern North America) [6]. The most known and studied species is Sambucus nigra L., which due to its variety of bioactive compounds, has been demonstrated to exhibit multiple beneficial effects [8,9]. At the present time, the elderberry crop has been introduced and also grown in many countries of the world, and the prospects for its use also depend on the interest of the public and the processing industry. Elderberry (Sambucus nigra L.) is cultivated in Central Europe, particularly in Romania, where it holds economic and agronomic importance. The plant thrives in the region’s temperate climate and fertile soils, making it ideal for large-scale farming. Romanian elderberry cultivation aligns with sustainable agricultural practices and meets the growing demand for its versatile applications. The flowers and fruits of elderberry are used by the food, cosmetic, and pharmaceutical industries, as well as in phytomedicine. The flowers are harvested and processed to produce herbal teas, which are widely appreciated for their health benefits, including their antioxidant and anti-inflammatory properties. These teas are a key component of natural remedies and wellness products. In addition to their flowers, elderberry fruits are utilized in the production of various food products, such as jams, juices, syrups, and dietary supplements, owing to their high content of vitamins, anthocyanins, and other bioactive compounds [10,11,12]. The effects demonstrated include cardiovascular protection [13], as well as antidiabetic [14], anti-tumour [15], antibacterial [16], antiviral [17,18], and anti-inflammatory [19,20] properties and antioxidant capacity [7,18,21]. The biological potential of various extracts derived from Sambucus nigra L. is closely linked to their chemical composition, which is influenced by factors such as cultivar, geographic location, ripening stage, and climatic conditions [7], and not least of all, by the extraction methods [22,23,24,25]. Generally, methanolic or aqueous fruit and flower extracts are rich in polyphenol as a plant secondary metabolite, classified into phenolic acids, flavonoids, anthocyanins, and lignans [20,23,26,27]. The chemical composition varies depending on the part of the plant used. The crude oils extracted from leaves are rich in fatty acids, including methyl linoleate and palmitic acid, along with the hydrocarbon tritetracontane and the aromatic compound benzaldehyde. The oil derived from flowers predominantly contains the hydrocarbons tritetracontane and n-hexatriacontane. These are followed by fatty acids and their derivatives, such as palmitic acid and linoleic acid, as well as alcohols, including 2-methyl-3,15-octadecadienol and 2-hexyl-1-octanol [28]. Instead, the essential oils (SNEO) obtained by hydrodistillation of Sambucus nigra L. reveal terpenes as their main class of compounds. Regarding the concentrations of terpenoids, Sambucus nigra L. essential oils are overall characterised by the predominates of different types of monoterpenes [28,29,30]. The elder fruit essential oils contain high amounts of β-damascenone, linalyl anthranilate, and linalool [29]. Those from flowers contain high concentrations of other monoterpenes, such as phelandrene, α- and γ-terpinene, terpinolene, safranal, carane, rose oxide, and epoxylinalol [28,29,30]. At the same time, the essential oils extracted from leaves are characterised by small amounts of monoterpenes, such as β-pinene and α-pinene [28].

Natural antioxidants, known as protective agents responsible for reducing the oxidative damage of organisms, are considered to be different phenolic constituents, i.e., flavonoids and terpenes. These compounds are found in variable concentrations in other plants, including Sambucus nigra L. The antioxidant capacity is based on their ability to interact with free radicals that initiate oxidation reactions or are produced during chain reactions upon the inhibition of oxidation processes, which reduces the activity of oxidase enzymes, or upon the complexation of transition metal ions, which catalyse oxidation reactions [31,32,33,34]. The berries and leaves of Sambucus nigra L. usually display a lower antioxidant activity than do the flowers [7,35] because elderflowers contain larger amounts of certain phenolic compounds (rutin, isoquercitrin, and astragalin) [34,36,37]. The antioxidant activity of elderflower essential oils is almost double that of leaf essential oils [28]. However, different chemical compositions in various regions imprint the antioxidant power. Gentscheva et al. [38] found that the extract from blossoms of Sambucus nigra L. from the Rhodope Region, Bulgaria, had the highest antioxidant activity. In contrast, the sample from the Dobrich Region has the lowest antioxidant activity. The difference is probably due to the higher altitude and lower temperature in the mountain regions, which influence the accumulation of secondary metabolites [38]. On the other hand, elderflower extract from lyophilised plant material demonstrated an antioxidant activity five times higher than that of the extract stabilised by freezing or using the air-drying method, since it contained from 96% to over 500% more polyphenols [39]. Thus, it is evident that various intrinsic and extrinsic factors are responsible for the efficacy of the antioxidant activity.

Concerning the antimicrobial activity of Sambucus nigra L., several previous studies demonstrated that the extracts displayed great activity against both Gram-positive and Gram-negative bacteria, respectively, particularly against the bacteria from the genera Staphylococcus, Streptococcus, Corynebacterium, Pseudomonas, Escherichia, Enterococcus, Klebsiella, Bacillus, and Proteus [40,41,42,43]. Instead, limited data are available regarding the antimycotic activity; the elderberry extracts demonstrated efficacy only against Candida albicans [44]. The ability of Sambucus nigra L. to inhibit or stop the growth and reproduction of microorganisms is attributed to the synergism between different phenolic acids and flavonoids rather than to certain compounds considered individually [41]. On the other hand, anthocyanins are the active compounds against Gram-negative bacteria [43].

While numerous studies have investigated the chemical composition and biological properties of various extracts [7,29,30,34,36,37], there remains limited knowledge about the compounds and bioactive potential of essential oil derived from Sambucus nigra L. The purpose of this research was to evaluate the antimicrobial activity of the essential oils against specific bacterial and fungal strains. This design was chosen to focus on the efficacy of the oils themselves and to explore their potential as natural antimicrobial agents and not as direct alternatives to existing pharmaceuticals.

The current study employed the computational in silico method in assessing the antimicrobial and antioxidant potential of the compounds identified in SNEO essential oil against target proteins. Molecular docking of the compounds with the proteins interfered with the action of the oil in regards to protein synthesis by blocking the tyrosyl-tRNA synthetase pathway (TyrRS) and peptidyl transferase activity, the protein prenylation pathway [45], and the plasma membrane fungal pathway [46], which would interfere with the microbial protein function [47,48,49,50]. Conversely, the free radical generation usually promoted by microbial infections would affect the catalase and glutathione peroxidase mopping activities, destabilising redox homeostasis and potentially altering cellular lipid peroxidation and DNA and protein expression [51].

2. Materials and Methods

2.1. Chemicals

The chemicals used included ethanol, 1,1-Diphenyl-2-Picrylhydrazyl, ascorbic acid, potassium hydroxide, hexane (Sigma-Aldrich Chemie GmbH, München, Germany), potassium hydrogen sulphate, and dimethyl sulfoxide (Geyer GmbH, Renningen, Germany).

2.2. Samples

The elderflowers were collected in May 2023 from a natural site in Dudestii Noi, 45°50′51″ N 21°06′30″ E, Timis County, Romania. The freshly harvested flower clusters were carefully selected, and any parts of the plant unsuitable for processing were removed, leaving only the superior plant material.

The plant vouchers (VSNH.ULST-BD72) were deposited in the botanical collection of the herbarium at the Botany Department, Faculty of Agriculture, King Michael I University of Life Sciences in Timisoara. The samples were air-dried under ambient conditions, with periodic turning until a constant mass was achieved. Once dried, the samples were stored in paper bags at 18–20 °C in the absence of light.

The essential oil was extracted from dried SN flowers using a Clevenger apparatus. A total of 30 g of flowers were placed in a distillation flask containing 300 mL of distilled water. The mixture was then boiled for 4 h. During this process, the vapours produced by the boiling were condensed, resulting in two phases, with the upper phase containing the essential oil. This oil was collected, poured into coloured glass vials, and stored at 4 °C. The essential oil yield (Y) was presented as a percentage and determined using the following formula:

$$\mathrm{Y}={\displaystyle \frac{\mathrm{M}\mathrm{e}}{\mathrm{M}\mathrm{p}}}\times 100$$

Y represents the percentage yield of essential oil, Me is the mass of the crucial oil in ml, and Mp is the mass of the plant material in grams [52].

AOAC method no. 2003.06 was used to ascertain the crude fat, which was determined using the Soxhlet method, using petroleum ether as an extraction solvent and Soxtest Raypa SX-6 MP equipment at 70 °C and a 60 min extraction [53]. A total of 5 g of pulverised flowers was introduced into the cartridges, and 50 mL of petroleum ether was used for each sample to extract lipids. The crude fat was collected, poured into coloured glass vials, and stored at 4 °C. The oil yield (Y_O) was presented as a percentage and determined using the following formula:

$${\mathrm{Y}}_{\mathrm{O}}=({\displaystyle \frac{\mathrm{M}\mathrm{o}}{\mathrm{M}\mathrm{p}}})\times 100$$

where Y_O represents the percentage yield of oil, Mo is the mass of the oil in grams, and Mp is the mass of the plant material in grams.

2.3. Fatty Acid Profile

Before gas chromatography (GC) analysis, the fatty acids in the oils were derivatised into methyl esters (FAMEs) using a 2M potassium hydroxide in a methanol solution. The oil was combined with 4 mL of hexane; then, 400 µL of a potassium hydroxide solution was added. The mixture was then vortexed for five minutes. Subsequently, 500 mg of potassium hydrogen sulphate was introduced. The organic phases were separated by centrifugation at 3000 rpm for 15 min and then transferred to vials for GC analysis. Fatty acid methyl esters (FAMEs) were identified using a Shimadzu GCMS QP 2010Plus instrument (Shimadzu Corporation, Tokyo, Japan), equipped with a mass spectrometer (MS) detector and an AT-Wax capillary column (30 m × 0.32 mm × 1 µm). The injection volume was 1.0 µL, with the injection port temperature set at 250 °C. Helium was used as the carrier gas at a flow rate of 1.8 mL/min, and a split ratio of 1:10 was applied.

The oven temperature was initially set at 110 °C and increased by 8 °C per minute until reaching 250 °C, where it was held for 7.5 min. The MS parameters included an ion source temperature of 210 °C and an interface temperature of 255 °C.

The identification and quantification of FAMEs were performed using the NIST05 library and the area normalization method. The results of the fatty acid (FA) analysis were expressed as a percentage of the total FAMEs.

2.4. GC–MS Volatile Compounds Profile

The essential oil of SN was analysed using gas chromatography–mass spectrometry (GC–MS) on a Shimadzu QP 2010 Plus instrument (Columbia, SC, USA), equipped with an AT-WAX capillary column (30 m × 0.32 mm × 1 µm). This was achieved by dissolving 20 µL of the essential oil in 1480 µL of hexane. From the resulting solution, a volume of 1 µL was injected into the GC apparatus at a temperature of 250 °C. The compounds in the hexane solutions were separated using an AT-5MS capillary column with dimensions of 30 m in length and 0.32 mm in diameter and a film thickness of 0.25 µm. The separation conditions were as follows: an initial temperature of 40 °C was maintained for two minutes, after which a temperature increase to 250 °C was initiated at a rate of 4 °C per minute, followed by an additional increase to 300 °C at a rate of 10 °C per minute, where it was maintained for five minutes. The mobile phase was helium 6.0, with a 1.92 mL/min flow rate. The interface temperature was set to 250 °C, while the ion source temperature was maintained at 210 °C. The separated compounds were identified through mass spectrometry, utilising MS scan mode acquisition (35–500 m/z) and the NIST database, while their quantification was performed using the normalised area method. The sample was injected in triplicate. A concordance of at least 90% was observed between the detected compounds and the database. The results were presented as a percentage of the total compounds identified. As previously described, the linear retention index (LRI) was calculated using the normal alkane RI for the same polar column [54].

2.5. Antioxidant Activity by 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) Assay

The antioxidant capacity of the essential oil was assessed using the DPPH method, following the procedure of Floares et al. [22]. A methanolic sample was prepared by dissolving 1 mL of EO in 10 mL of methanol (Sigma–Aldrich; Merck KGaA, Darmstadt, Germany), and the extracts were stored at 2–4 °C until analysis. Different concentrations (2.00, 4.00, 6.67, 10.00, and 20.00 mg/mL) were prepared by diluting the base extract. For each dilution, 3 mL was mixed with 1 mL of 0.3 mM DPPH (Sigma-Aldrich, Taufkirchen, Germany) solution and incubated for 30 min in the dark. The absorbance of the samples was measured at 517 nm using a UV–VIS spectrophotometer (Specord 205; Analytik Jena AG, Jena, Germany). Each sample was tested in triplicate, and the average was recorded.

A control sample was analysed simultaneously, in which the extract was replaced with methanol, while maintaining the same volume and concentration of DPPH solution. Ascorbic acid (0.006–0.016 mg/mL in methanol) from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany) was used as a positive control.

The antioxidant activity was determined as the percentage of radical scavenging activity (RSA) using the following Formula (1):

$$\mathrm{RSA}(\%)=({\displaystyle \frac{{\mathrm{A}}_{\mathrm{control}}-{\mathrm{A}}_{\mathrm{sample}}}{{\mathrm{A}}_{\mathrm{control}}}})\times 100$$

(1)

where A_control = the absorbance value of the control sample, and A_sample = the absorbance value of the essential oil sample.

The antioxidant capacity of the sample was expressed as the IC_{50 value} and was compared to ascorbic acid.

2.6. Evaluation of the Antimicrobial Activity

In this study, the antimicrobial activity of SNEO was evaluated using a broth microdilution method against a range of Gram-positive and Gram-negative ATCC strains. The selection of bacterial and fungal strains in this study was guided by several important considerations. The inclusion of both Gram-positive and Gram-negative bacteria enables the evaluation of the broad-spectrum antimicrobial potential of S. nigra essential oil. This accounts for the structural and physiological differences between these bacterial groups, which can influence their susceptibility to natural antimicrobial agents. The bacterial strains include both common nosocomial pathogens (Pseudomonas aeruginosa, Staphylococcus aureus) and foodborne pathogens (Listeria monocytogenes, Salmonella typhimurium), covering various ecological niches and infection sources. The inclusion of Clostridium perfringens represents the anaerobic pathogens, broadening the scope of the study. Testing Candida albicans and Candida parapsilosis addresses the potential antifungal properties of SNEO, targeting two fungal species often implicated in opportunistic infections. The Gram-positive ATCC strains included Streptococcus pyogenes (ATCC 19615), Staphylococcus aureus (ATCC 25923), C. perfringens (ATCC 13124), Listeria monocytogenes (ATCC 19114), and Bacillus cereus (ATCC 10876). The Gram-negative strains tested were Pseudomonas aeruginosa (ATCC 27853), Shigella flexneri (ATCC 12022), Escherichia coli (ATCC 25922), Salmonella typhimurium (ATCC 14028), and Haemophilus influenzae tip B (ATCC 10211). Two Candida species were selected as fungal representatives: Candida parapsilopsis (ATCC 22019) and Candida albicans (ATCC 10231). All ATCC strains were sourced from the culture collection of the Laboratory of Microbiology at the Interdisciplinary Research Platform, King Michael I University of Life Sciences, Timisoara.

Following a method described in prior research [1,3], a stock solution of each essential oil was prepared in dimethyl sulfoxide (DMSO). To perform the test, 50 µL of the stock solution was added to 100 µL of a freshly prepared bacterial suspension, standardized to McFarland standard optical density of 0.5 (1.5 × 10⁸ CFU/mL), followed by serial dilutions.

SNEO was tested at concentrations of 0.1 mg/mL, 0.125 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1 mg/mL, 2 mg/mL, 4 mg/mL, 8 mg/mL, 16 mg/mL, and 32 mg/mL. A bacterial culture in brain heart infusion (BHI) broth, without the essential oil, served as the positive control.

The minimum inhibitory concentration (MIC), defined as the lowest concentration at which no visible bacterial growth was observed, was determined spectrophotometrically by measuring the optical density [1,2,3]. The results were interpreted by calculating two indicators, bacterial growth rate (BGR) and bacterial inhibition rate (BIR), using Formulas (2) and (3), as follows:

BGR = OD_sample/OD_{negative control} × 100 (%)

(2)

BIR = 100 − BGR (%)

(3)

* where OD_sample refers to the optical density of the sample, which contains a specific type and concentration of essential oils being tested, measured at 540 nm as the average of triplicate readings, and OD_{negative control} represents the optical density at 540 nm, calculated as the average of triplicate readings for the selected bacteria in BHI broth.

2.7. Molecular Docking Studies

PyRx-Python Prescription 0.8 was used in the docking assessment between the most abundant compounds (≥1%) identified in SNEO and the antimicrobial and antioxidant protein targets (PDB ID: 1JIK, 6G9S, 8JZN, 3DRA, 2CAG, 2P31). The SDF formats of the compounds were downloaded from https://pubchem.ncbi.nlm.nih.gov/ (accessed on 9 November 2024) [55], while the PDB formats of the proteins were retrieved from https://www.rcsb.org/ (accessed on 8 November 2024) [56].

The coordinates of the grid box, as presented in

Table 1. Binding interaction coordinates for the proteins in the molecular docking.

S/No	Proteins	PDB ID	Interaction Coordinates
1	Tyrosyl-tRNA synthetase	1JIK	Center X: 34.9074, Y: 11.9032, Z: 89.6565 Dimensions (Angstrom) X: 69. 6981, Y: 43.8698, Z: 51.0095
2	Penicillin-binding protein 2	6G9S	Center X: 29.4505, Y: 55.2099, Z: 39.0797 Dimensions (Angstrom) X: 82.3524, Y: 88.9743, Z: 52.0756
3	Catalase	2CAG	Center X: 63.3670, Y: 18.0277, Z: 16.2820 Dimensions (Angstrom) X: 63.6866, Y: 77.0070, Z: 90.1406
4	Glutathione peroxidase	2P31	Center X: −8.2367, Y: −0.6748, Z: −23.6360 Dimensions (Angstrom) X: 37.8548, Y: 34. 5011, Z: 79.1468
5	1,3-β-glucan synthase	8JZN	Center X: 157.249, Y: 160.457, Z: 147.4421 Dimensions (Angstrom) X: 90.7434, Y: 105.0962, Z: 121.5736
6	Protein geranylgeranyltransferase-I	3DRA	Center X: 28.7147 Y: 41.6649 Z: 20.5575 Dimensions (Angstrom) X: 83.9663, Y: 709289, Z: 64.3607

, were applied to commence the docking of the compounds and each of the proteins. Post-docking analysis was performed in Discovery Studio Visualizer v21.1.0.20298 (BIOVIA, San Diego, CA, USA), which allowed for the 3D and 2D visualisation of the binding interactions. The ligand–protein interactions involving hydrophobic and hydrophilic bonds were collected and used to establish the predictable pharmacological profile between the compounds of high abundance and the respective microbial proteins.

2.8. Statistical Analysis

Statistical analysis was carried out using JASP 0.19. Descriptive statistics, including the mean and standard deviation, were computed to assess the study data. Group comparisons were performed using analysis of variance (ANOVA), followed by Tukey’s post hoc test. A significance level of p < 0.05 was considered statistically significant.

3. Results

3.1. Fatty Acid Profile

Subsequent to a Soxhlet extraction with a yield (w/v) of 1.46 ± 0.08, the GC–MS analysis identified ten fatty acids in the crude fat extracted from elderflower, seven being saturated fatty acids and representing 45% of the total (

Table 2. Fatty acids profile from crude fat of elderflower analysed by GC–MS.

No. crt	Fatty Acid as Methyl Ester	Percentage of Total Compounds (%)
1	Palmitic acid C16:0	26.574 ± 0.68
2	Linolenic acid C18:3 Δ9,12,15 (Z,Z,Z)	12.794 ± 0.18
3	Linoleic acid C18:2, Δ9,12(Z,Z)	9.397 ± 0.62
4	Oleic acid C18:1, Δ9 (Z)	32.806 ± 0.82
5	Stearic acid, C18:0	3.996 ± 0.28
6	Arachidic acid, C20:0	1.025 ± 0.01
7	Behenic acid, C22:0	1.944 ± 0.13
8	Lignoceric acid, C24:0	4.554 ± 0.26
9	Cerotic acid, C26:0	4.217 ± 0.50
10	Montanic acid, C28:0	2.690 ± 0.37
	Saturated Fatty Acids (SFA)	45.002 ± 0.31
	Monounsaturated Fatty Acids (MUFA)	32.806 ± 0.82
	Polyunsaturated Fatty Acids (PUFA)	22.191 ± 0.39

).

3.2. GC–MS Volatile Compounds Profile

The essential oil obtained produced an oil yield of (w/v): 0.14 ± 0.01%, and the GC–MS analysis revealed 25 compounds, representing chemical constituents present by more than 0.5% in the composition of SNEO, as detailed in

Table 3. Volatile chemical constituents of SNEO analysed by GC–MS.

No. Crt	Compounds	Percentage of Total Compounds (%)	RI c/ Rir
1	1,4-Hexadiene, 3,3,5-trimethyl-	1.82 ± 0.109	960/964
2	β-Linalool	1.56 ± 0.052	1086/1089
3	1,5,7-Octatrien-3-ol, 3,7-dimethyl-	1.55 ± 0.053	1090/1089
4	Nonanal	4.36 ± 0.069	1098/1095
5	cis-Rose oxide	3.87 ± 0.070	1110/1112
6	trans-Rose oxide	1.74 ± 0.009	1129/1128
7	Nerol oxide	0.51 ± 0.008	1150/1144
8	Dihydroedulan II (cis)	0.50 ± 0.010	1280/1282
9	Edulan I, dihydro-	1.83 ± 0.005	1320/1318
10	6,8-Nonadien-2-one, 6-methyl-5-(1-methyl ethylidene)-	1.02 ± 0.082	1380/1382
11	2-Pentadecanone, 6,10,14-trimethyl-	1.96 ± 0.191	1801/1804
12	Heptadecane	7.96 ± 0.123
13	2,6-Octadiene, 2,6-dimethyl-	0.57 ± 0.048	1950/1948
14	Octadecane	1.14 ± 0.275
15	1-Octadecanol	0.59 ± 0.035	2085/2090
16	Nonadecane	23.19 ± 0.409
17	Eicosane	2.94 ± 0.051
18	9-Tricosene, (Z)-	0.44 ± 0.055	2270/2274
19	Heneicosane	25.08 ± 0.498
20	Docosane	2.06 ± 0.007
21	9-Hexacosene	1.07 ± 0.065	2574/2570
22	Tetracosane	9.35 ± 0.126
23	Pentacosane	2.34 ± 0.112
24	Squalene	1.95 ± 0.092	2835/2833
25	Tetratriacontane	0.50 ± 0.008
	Total compounds	100	100
	Terpene hydrocarbons	1.95 ± 0.092
	Oxygenated terpene	11.59 ± 0.018
	Alcohols	2.14 ± 0.054
	Aldehydes and ketones	7.35 ± 0.076
	Alkanes	74.59 ± 0.116
	Other (unsaturated hydrocarbons)	2.38 ± 0.094

.

3.3. Antioxidant Capacity by 1,1-Diphenyl-2-Picrylhydrazyl (DPPH) Assay

Antioxidant activity represents a fundamental biological property with significant implications for various industries, including cosmetics, food, and beverages. Since antioxidant potential is a critical measure for determining the therapeutic advantages of plants, antioxidant assays were employed in this study, specifically the radical scavenging (DPPH) method. This specific assay is frequently recommended due to its simplicity, rapidity, reproducibility, and cost-effectiveness, which collectively make it an optimal tool for evaluating the antioxidant activity of plants.

To assess the radical scavenging activity using the DPPH method, five concentrations (20.00 mg/mL, 10.00 mg/mL, 6.67 mg/mL, 4.00 mg/mL, and 2.00 mg/mL) of essential oil dissolved in methanol were prepared from the three samples tested. At the same time, the antioxidant activity of five ascorbic acid solutions at varying concentrations (0.006–0.016 mg/mL) was measured as a positive control, with the highest concentration (0.016 mg/mL) exhibiting 91.13% inhibition (Table 3).

The IC₅₀ was calculated and expressed in mg/mL (

Table 4. The DPPH radical scavenging activity (% inhibition) of essential oil vs. ascorbic acid.

Samples		Acid Ascorbic
Concentration (mg/mL)	SN % Inhibition	Concentration (mg/mL)	% Inhibition
2.00	17.61 ± 0.06	0.006	23.81 ± 0.03
4.00	37.97 ± 0.06	0.008	41.73 ± 0.06
6.67	69.81 ± 0.07	0.010	55.47 ± 0.05
10.00	82.47 ± 0.06	0.014	79.16 ± 0.08
20.00	85.38 ± 0.03	0.016	91.13 ± 0.06

). The IC₅₀ is the concentration of essential oil required to induce 50% DPPH inhibition.

The results are presented as the mean of three determinations ± standard deviation (SD).

The percentage of DPPH inhibition was 85.38% at 20 mg/mL, 82.47% at 10 mg/mL, 69.81% at 6.67 mg/mL, 37.97% at 4 mg/mL, and 17.61% at 2 mg/mL.

As shown in Table 4, the highest concentration (20 mg/mL) exhibited the maximum radical scavenging activity.

The percentage of DPPH inhibition remained elevated at two lower concentrations (10 mg/mL and 6.67 mg/mL). However, it decreased significantly at the lowest concentration tested (2 mg/mL).

As shown in

Table 5. The IC₅₀ value of the essential oil sample vs. ascorbic acid.

Samples	SN	Ascorbic Acid
IC50 ± SEM	2.520 ± 0.082 a	2.525 ± 0.014 a
R2	0.9213	0.9919
Hill Slope	18.004	17.207

The results are presented as the mean of three determinations ± standard deviation (SD). (^a) The mean differences between SN and ascorbic acid were compared using the ANOVA test; values with the same superscript are not statistically different (p > 0.05).

, the IC₅₀ value for SN is lower than that of ascorbic acid, suggesting that SN may offer more significant protection against oxidation. Statistical analysis indicated no significant difference between ascorbic acid and SN essential oil (p = 0.927), suggesting that the antioxidant effect of SN essential oil may be comparable to that of ascorbic acid.

3.4. Evaluation of the Antimicrobial Activity

Table 6. Bacterial inhibition rates of SNEO against Gram-positive ATCC strains.

SNEO mg/mL	S. pyogenes	S. aureus	L. monocytogenes	B. cereus	C. perfringens
0.1	−36.61	−61.59	62.79	57.25	−39.25
0.125	−19.73	−48.57	53.85	56.39	−7.28
0.25	−6.87	−1.78	6.52	51.52	5.23
0.5	10.28	−1.78	6.57	43.56	17.59
1	15.26	8.33	5.82	38.80	20.39
2	17.06	14.99	2.27	16.07	22.70
4	22.04	16.52	−0.47	−1.57	32.76
8	30.20	31.80	−7.75	−10.39	38.62
16	35.18	39.45	−12.20	−10.88	45.44
32	42.23	39.99	−47.38	−57.79	59.21
IC50 mg/mL	10.40	9.84	1.93	3.15	8.92

presents the antimicrobial activity of SNEO against ATCC strains, including Gram-positive bacteria, and

Table 7. Bacterial inhibition rates of SNEO against Gram-negative bacteria and Candida spp. ATCC strains.

SNEO mg/mL	P. aeruginosa	S. lexneri	E. coli	S. typhimurium	H. influenzae	C. parapsilosis	C. albicans
0.1	33.37	−6.45	−6.50	−19.37	−18.70	−25.27	73.78
0.125	30.08	−7.80	−6.21	−17.62	−16.35	−22.95	72.37
0.25	28.09	−9.68	−5.54	−17.57	−13.07	−5.61	59.96
0.5	25.90	−11.24	−4.51	−11.68	−10.84	−4.73	24.21
1	21.41	−13.74	−3.47	−9.03	−7.19	−1.39	13.73
2	15.04	−15.71	−2.59	−3.42	−4.74	2.78	12.59
4	8.96	−16.55	−1.03	−1.82	−2.58	5.70	11.40
8	2.59	−18.21	−0.67	9.37	−1.14	14.46	10.08
16	0.50	−19.46	−0.14	21.48	−0.97	34.26	4.24
32	−8.27	−22.16	3.25	26.85	4.60	43.30	−24.90
IC50 mg/mL	11.73	9.65	2.90	8.85	8.72	12.07	3.08

shows the activity against Gram-negative bacteria and two fungal Candida strains.

In evaluating the antibacterial activity of SNEO against Gram-positive bacteria (Table 6), the efficacy was generally low, as the inhibition rates were negative across all strains tested. However, S. pyogenes, S. aureus, and C. perfringens displayed an ascending trend in regards to antibacterial activity, with inhibition positively correlated to increased oil concentrations. The MIC values for these strains were 0.25 mg/mL for C. perfringens, 0.5 mg/mL for S. pyogenes, and 1 mg/mL for S. aureus. In contrast, the activity of SNEO against L. monocytogenes and B. cereus showed no positive inhibition trend. Instead, the bacterial inhibition rate (BIR%) decreased as the concentration increased, resulting in a strain-boosting effect, an effect demonstrated by a bacterial growth rate higher by 50% compared to the strain unaffected by the oil activity. Compared to the untreated strains, L. monocytogenes showed a negative inhibition rate of −47.38%, and B. cereus showed an even greater negative inhibition rate of −57.79%, indicating a growth-promoting effect at higher oil concentrations.

The following table represents the antimicrobial inhibitory activity of SNEO against different microbial strains, expressed as percentage inhibition, at various concentrations of mg/mL, along with their IC₅₀ values.

In most cases, the inhibition is concentration-dependent, that is to say, an enhanced SNEO concentration results in higher antimicrobial activity; on the other hand, some strains, for example, S. flexneri and P. aeruginosa, exhibit negative percent values at high concentrations. SNEO shows moderate activity against P. aeruginosa, where the inhibition percentage is reduced with an increase in concentration. The IC₅₀ value is 11.73 mg/mL; hence, it is moderately susceptible. The IC₅₀ value for S. flexneri is 9.65 mg/mL, which reveals that it has better sensitivity than does P. aeruginosa. Inhibition increases in a dose-dependent manner. Regarding E. coli, the strain presented an intermediate sensitivity, with an IC₅₀ value of 2.90 mg/mL; however, SNEO showed potent inhibition at concentrations much lower than the respective IC₅₀, indicating a strain-boosting effect. The strains of S. typhimurium and H. influenzae are moderately susceptible, with IC₅₀ values of 8.85 and 8.72 mg/mL, respectively. The inhibition is dose-dependent but much less pronounced than that for E. coli. SNEO exhibits potent activity against C. parapsilosis, with an IC₅₀ value of 12.07 mg/mL. C. albicans is the most sensitive tested organism, with an IC₅₀ value of 3.08 mg/mL. Inhibition is high even at the lowest concentration tested (73.78% at 0.1 mg/mL), suggesting an exceptional antifungal potency. SNEO exhibits more activity against fungal strains, especially C. albicans, than against bacterial strains, which might be attributed to differences in the composition and permeability of the cell wall between fungi and bacteria. Among the bacteria, E. coli is the most susceptible to SNEO, while P. aeruginosa shows moderate resistance, in agreement with its known stronger membrane structure and efflux mechanisms.

3.5. Molecular Docking

In the present study,

Table 8. Docking scores of the most abundant compounds (ligands) and their binding interactions with microbial (1JIK, 6G9S, 8JZN, 3DRA) and oxidant proteins (2CAG, 2P31).

S/No	Ligands	PubChem ID	Binding Energy (Kcal/mol)						Interactions: Hydrophobic and Hydrophilic
			1JIK	6G9S	8JZN	3DRA	2CAG	2P31	1JIK	6G9S	8JZN	3DRA	2CAG	2P31
1	Nonanal	31289	−4.5	−4.4	−4.8	−4.6	−4.9	−3.5	Hydrophobic: Leu128, Ile131, Phe136, Leu173	Hydrophobic: Arg68, Lys162, Val179 Hydrophilic: His203, Asp204	Hydrophobic: Ile1655, Phe1658, Val1745, Phe1811, Cys1814 Hydrophilic: His1654	Hydrophobic: Phe37, Met164, Trp300, Leu352 Hydrophilic: Tyr163, Thr375	Hydrophobic: Arg51, Ala112, Val125, Phe313, Tyr337, Ala340, His341 Hydrophilic: Ser93, Gly110	Hydrophobic: Val38, Pro113, Phe115Hydrophilic: Asn32
2	cis-Rose oxide	1712087	−5.3	−6.0	−6.0	−5.9	−6.3	−5.4	Hydrophobic: Cys37, Ala39, Leu46, Pro53, Phe54, Ile103 Hydrophilic: His50	Hydrophobic: Ala65, Arg68, Tyr161, Lys162, Val179, His203 Hydrophilic: Pro66	Hydrophobic: Ile713, Ile715, Phe821 Hydrophilic: Arg1182, Gln1376	Hydrophobic: Phe37, Arg160, Tyr163, Met164, Cys225, Trp300, Leu352	Hydrophobic: Arg51, Arg52, Ala112, Val125, Phe313, Tyr337, Ala340	Hydrophobic: Phe103, Arg106, Arg106
3	trans-Rose oxide	7093102	−5.4	−5.9	−6.1	−6.7	−6.2	−4.5	Hydrophobic: Leu128, Ile131, Leu133, Phe136, Leu173	Hydrophobic: Ala65, Arg68, Tyr161, Lys162, Val179	Hydrophobic: His1654, Ile1655, Phe1658, Val1745	Hydrophobic: Pro220, His249, Val252	Hydrophobic: Ala112, Val125, Phe313, Tyr337, Ala340, His341 Hydrophilic: Arg51	Hydrophobic: Lys130, Ala133
4	Edulan I, dihydro-	521066	−5.9	−6.9	−7.0	−7.6	−5.9	−5.3	Nil	Hydrophobic: Tyr161, Arg163, His203	Nil	Nil	Nil	Nil
5	2-Pentadecanone, 6,10,14-trimethyl-	10408	−5.2	−5.9	−6.5	−5.5	−7.0	−4.5	Hydrophobic: Ile78, Ile131, Phe136, Tyr165	Hydrophobic: Ala65, Arg68, Arg164, Val179	Hydrophobic: Tyr1451, Ala1454, Arg1684, Phe1687, Ala1742, Leu1746	Nil	Hydrophobic: Phe132, Phe140, His197, Leu278, Arg333, Tyr337, Ala340 Hydrophilic: Arg52, Omt53	Hydrophobic: Arg34, Val38, Pro113, Phe115
6	Heptadecane	12398	−4.9	−5.0	−5.5	−5.6	−6.4	−4.8	Hydrophobic: Leu128, Ile131, Leu133, Phe136, Leu173	Hydrophobic: Ala65, Arg68, Tyr161, Arg164, Val179, Ala201	Hydrophobic: Tyr1451, Ala1454, Arg1455, Ile1680, Arg1684, Ala1742, Leu1746	Hydrophobic: Phe37, Phe99, Arg160, Tyr163, Met164, His219, Phe222, Cys225, Trp300, Leu352	Hydrophobic: Phe132, Phe140, His197, Leu278, Arg333, Tyr337, Ala340, His341	Hydrophobic: Arg34, Lys98, Phe103, Arg106
7	Nonadecane	12401	−5.0	−3.7	−6.1	−4.7	−6.6	−4.0	Hydrophobic: Met77, Leu128, Ile131, Phe136, Leu173	Hydrophobic: Leu352, Pro355, Trp357, Trp358, Pro361	Hydrophobic: His1654, Ile1655, Phe1658, Ala1742, Val1745, Leu1746, Val1749, Phe1811, Cys1814	Hydrophobic: Tyr67, Trp106, Pro140, His145	Hydrophobic: Arg51, His54, Ala112 Val125 Pro137, Phe140, Phe313, Met329, Arg333, Tyr337, Ala340, His341	Hydrophobic: Lys98, Arg105, Arg106
8	Eicosane	8222	−5.0	−4.8	−5.5	−5.6	−6.7	−3.2	Hydrophobic: Ile78, Leu128, Ile131, Leu133, Phe136, Leu173	Hydrophobic: Ala65, Arg68, Tyr161, Lys162, Ala201	Hydrophobic: His1654, Ile1655, Phe1658, Val1676, Ile1680, Ala1742, Val1745, Phe1811, Ile1815	Hydrophobic: Phe37, Leu98, Phe99, Arg160, Tyr163, Met164, Leu167, His219, Phe222, Cys225, Trp300	Hydrophobic: Arg51, Phe132, Phe140, His197, Leu278, Met329, Arg333, Tyr337, Ala340	Hydrophobic: Arg106, Phe103
9	Heneicosane	12403	−5.1	−5.4	−4.8	−5.0	−6.0	−3.3	Hydrophobic: Ile78, Leu128, Ile131, Leu133, Phe136, Leu137, Tyr165, Leu173	Hydrophobic: Ala65, Arg68, Lys159, Tyr161, Lys162, Arg164, Val179	Hydrophobic: Met458, Ile578, Tyr622, Val626, Phe629, Tyr633, Val1284, Leu1288	Hydrophobic: Ala33, Tyr36, Phe37, Leu98, Phe99, Pro140, Arg160, Val161, Tyr163, Met164, Cys225, Met348	Hydrophobic: Arg52, Phe132, Phe140, Pro141, His197, Leu278, Arg333, Tyr337, Ala340	Hydrophobic: Arg105, Arg106
10	Docosane	12405	−4.9	−4.9	−5.6	−5.2	−6.4	−4.1	Hydrophobic: Leu128, Ile131, Phe136, Leu173	Hydrophobic: Ala65, Arg68, Tyr161, Lys162, Arg164, Val179, His203	Hydrophobic: Ile1507, Leu1510, Val1651, Ile1652, Ile1655, Phe1744, Val1807	Hydrophobic: Tyr67, His145	Hydrophobic: Arg51, Arg52, His54, Ala112, Val125, Pro137, Phe140, Phe313, Met329, Arg333, Tyr337, Ala340, His341	Hydrophobic: Lys98, Arg105, Arg106
11	Tetracosane	12592	−4.3	−4.8	−6.3	−4.0	−5.7	−4.4	Hydrophobic: Cys37, Ala39, His50, Pro53	Hydrophobic: Ile64, Ala65, Arg68, Tyr161, Lys162	Hydrophobic: Tyr1451, Ala1454, Arg1455, Ile1680, His1654, Ile1655, Phe1658, Ala1741, Ala1742, Val1745, Leu1746, Phe1811	Hydrophobic: Leu20, Pro21, Ala24, Ile34, Pro347, Met348, His349	Hydrophobic: Arg51, Arg52, His54, Ala112, Val125, Phe313, Tyr337, Ala340, Tyr343, Arg344	Hydrophobic: His63, Arg65, Ala66, Leu70, Val165
12	Pentacosane	12406	−4.9	−3.9	−5.4	−5.0	−5.9	−3.4	Hydrophobic: Ile78, Arg125, Leu128, Ile131, Phe136, Leu173	Hydrophobic: Leu352, Phe353, Pro355, Trp358, Pro361	Hydrophobic: Phe463, Trp464, Ala468, Val502, Ile681, Ala682, Phe685	Hydrophobic: Tyr36, Phe37, Tyr67, Phe99, Arg160, Tyr163, Met164, Trp300, Met348	Hydrophobic: Arg51, Arg52, Pro137, Phe140, Pro141, Met329, Arg333, Tyr337, Ala340	Hydrophobic: Phe103, Arg105, Arg106
13	Squalene	638072	−6.3	−4.9	−7.7	−6.1	−6.9	−6.5	Hydrophobic: Leu128, Phe136, Leu173	Hydrophobic: Leu352, Pro355, Trp357, Trp358, Pro361	Hydrophobic: Met458, Ile462, Met465, Tyr466, Tyr469, Ile578, Phe629, Tyr633, Tyr637, Leu1288	Hydrophobic: Tyr67	Hydrophobic: Phe132, Pro137, Leu138, Phe140, Pro141, Arg333	Hydrophobic: Lys98, Phe103, Arg106

showed a keen docking association between the macromolecular targets involved in defining new antibacterial agents, representing attractive target enzymes for antioxidant activity, as put forth by the identified compounds in SNEO. The molecular docking score of the protein–ligand revealed interesting binding energies ranging from −7.0 to −3.2 kcal/mol, indicating the relative binding affinity in the docked complex. Accordingly, the current study showed cis-rose oxide (−5.3 kcal/mol with 1JIK; −6.0 kcal/mol with 8JZN), nonanal (−4.4 kcal/mol with 6G9S; −4.8 kcal/mol with 8JZN; -4.6 kcal/mol with 3DRA; −4.9 kcal/mol with 2CAG; −3.5 kcal/mol with 2P31), 2-Pentadecanone, 6,10,14-trimethyl- (−7.0 kcal/mol with 2CAG), and tans-rose oxide (−6.2 kcal/mol with 2CAG) exhibited predictable binding to the pockets of the respective protein targets (Figure 1).

In the binding site of tyrosyl-tRNA synthetase, cis-rose oxide was involved in hydrogen bonding (His50), carbon–hydrogen bonding (Ile103), and other alkyl–hydrophobic bonding (Figure 1a). Similarly, nonanal was observed to occupy the penicillin-binding protein 2 binding pocket in hydrogen-bonding (His203, Asp204) and alkyl interactions (Arg68, Lys162, Val179). At the same time, cis-rose oxide showed a carbon–hydrogen bond (Pro66) and other alkyl bonds with the same protein (Figure 1b,c). As for the binding complex of catalase, 2-Pentadecanone, 6,10,14-trimethyl- was observed to display a hydrogen-bond interaction (Arg52, Omt53), nonanal had hydrogen-bond interaction (Ser93), carbon-hydrogen bond (Gly110); trans-rose oxide was also involved in hydrogen-bonding (Arg51), all of which were among alkyl and sigma hydrophobic bonds (Figure 1d–f). Furthermore, in the binding pattern of glutathione peroxidase, nonanal showed hydrogen-bonding interaction (Asn32) and other alkyl/sigma hydrophobic bonds (Figure 1g). Additionally, the possible interactions between 1,3-β-glucan synthase and nonanal are hydrogen-bond formations (His1654), among other hydrophobic alkyl and pi–alkyl bonds; likewise cis-rose oxide interacts with the protein in both hydrogen (Arg1182), carbon–hydrogen (Gln1376), and other hydrophobic bonds (Figure 1h,i). The best binding geometry of protein geranylgeranyltransferase-I was recorded with nonanal, involving hydrogen bonds with two amino acid residues (Tyr163, Thr375) and four alkyl–/pi–alkyl bonds (Figure 1j).

4. Discussion

The chemical composition of Sambucus nigra L. varies due to several factors, including climatic conditions, geographic location, cultivar, ripening stage, and the specific part of the plant [7,28,29]. Additionally, the various extraction methods can also produce different chemical profiles from natural products [7,22,38]. Knowing that chemical compounds determine biological properties, it is crucial to analyse the obtained natural products thoroughly. Regarding Sambucus nigra L. essential oil (SNEO), only a few studies described its compounds and biological aspects, especially the antioxidant properties [28,29].

Among the chemical compounds of SN oil, the fatty acids are presented in different concentrations and varying principal compounds. Szymański et al. [28] reported a total % of fatty acids and derivatives of 9.14%, with palmitic acids as the main compound (43.47%). Instead, the SN oil from the present study is dominated by oleic acid (32.806%), a monounsaturated fatty acid (MUFA). Oleic acid has been primarily recognised for its anti-inflammatory and cardioprotective effects, attributed to its ability to regulate cholesterol levels and promote vascular function [57,58,59,60]. Other fatty acids, such as linolenic acid (12.794%) and linoleic acid (9.397%), represent the major PUFA (polyunsaturated fatty acids) components producing the oil’s antioxidant and anti-inflammatory properties [61,62,63]. Moreover, the studied SN oil contains a significant concentration of palmitic acid (26.574%), a saturated fatty acid (SFA) that enhances oil stability and plays a crucial role as a fundamental element in energy storage and skin barrier integrity [64,65,66,67]. Other SFAs, such as stearic acid (3.996%) and lignoceric acid (4.554%), contribute to the moisturising and emollient characteristics, making it useful for cosmetic applications [68]. A high amount of saturated fatty acids (45.002%) provides structural stability, while the monounsaturated (32.806%) and polyunsaturated fatty acids (22.191%) perhaps explain part of the biological activities. These results present the balance of the SN oil components.

The GC–MS profile of elderflower essential oils reveals different bioactive compounds in various concentrations. Vujavonic et al. [29] showed that the essential oil obtained from the traditionally dried elderflower contained carane as a major compound (13.91%), followed by α-limonene diepoxide (7.23%), methyl salicylate (7%), and caryophyllene (6.55%). Szymański et al. [28] demonstrated that the essential oils are dominated by tritetracontane (20.237%). Other principal compounds included 2-Methyl-3,13-octadecadienoic (5.438%), 9,10-Epoxyoctadecan-1-ol (4.202%), and rose oxide (3.089%) [28]. Our research demonstrated that the most abundant compounds were alkanes (74.59%), heneicosane (25.08%), and nonadecane (23.19%). Other significant constituents include tetracosane (9.35%) and heptadecane (7.96%). The alkanes are known for their potential role in enhancing the stability of oils due to their hydrophobic interactions [69,70]. Among the terpenes class, cis-rose oxide (3.87%), trans-rose oxide (1.74%), and β-Linalool (1.56%) were distinguished. These results are similar to those demonstrated by Szymański et al. [28], which showed that rose oxide was the main compound in the monoterpenes group. It is well documented that the biological properties of oxygenated and alcohol terpenes consist of high antimicrobial and antioxidant activities, likely attributed to their interaction with microbial enzymes and their mechanisms of oxidation [3,71,72,73,74].

The potential of elderflower extract as an antioxidant has attracted considerable attention, leading to numerous investigations into its effectiveness [20,23,26,27,36,38]. However, the antioxidant properties of the essential oil obtained from the flowers have not been extensively researched until now. The present study highlighted that SNEO had an IC₅₀ value of 2.520 mg/mL, similar to that obtained using ascorbic acid. Similar values (225.40 µL/mL) were also noted by Szymański et al. [28] when they studied the essential oil obtained from Sambucus nigra L. flowers via hydrodistillation, using essential oil from Matricaria recutita flowers as a positive control (90.78 µL/mL).

In terms of antimicrobial properties, various extracts of Sambucus nigra L. have been shown to exhibit strong antibacterial activity against both Gram-positive and Gram-negative bacteria [31,36,37,38] but also against various classes of fungus [30,44]. To our knowledge, this is the first study regarding SNEO antimicrobial activity. According to our results, all the Gram-positive bacterial strains were sensitive to SNEO, with IC₅₀ values between 1.93 mg/mL and 10.40 mg/mL. The essential oil showed moderate activity against S. pyogenes (IC₅₀ = 10.40 mg/mL) and S. aureus (IC₅₀ = 9.84 mg/mL), with a positive trend as the concentration grew. Also, C. perfringens presented a moderate sensitivity to SNEO, with an IC₅₀ of 8.92 mg/mL. L. monocytogenes was the most sensitive bacteria to the SNEO, while S. aureus was the most resistant, requiring a 1 mg/mL concentration to inhibit its growth. Compared to Gram-positive bacteria, Gram-negative bacteria were more resistant, with IC values between 2.90 mg/mL and 11.73 mg/mL. This can be explained by the difference in the cell envelope structure between the two groups of bacteria. The Gram-negative type contained an outer lipopolysaccharide membrane [75]. Of all the studied Gram-negative bacteria, E. coli was the most sensitive (IC₅₀-2.90 mg/mL). The antifungal activity of SNEO was evident, especially against C. albicans, the most sensitive fungal culture, with a MIC value of 0.1 mg/mL and an IC₅₀ of 3.08 mg/mL.

SNEO revealed a dose-dependent increase in antimicrobial activity, which means that it exhibits increased activity at higher concentrations. The broad-spectrum efficacy of SNEO is attributed to its chemical compounds, whose mechanisms of action are entirely elucidated. The high content of fatty acids, such as palmitic acid and oleic acid, may disrupt microbial cell membrane integrity. Still, it has been demonstrated to promote membrane fluidity, which can facilitate microbial cell lysis [76,77,78,79]. A similar effect seems to be attributed to nonanal and rose oxides. Cis- and trans-rose oxides have been reported to exhibit strong antifungal and antibacterial effects, likely through interactions with microbial enzymes and membrane structures [80]. Similarly, β-Linalool (1.56%), a terpene alcohol, is known for its antimicrobial and anti-inflammatory activity achieved through membrane disruption by inhibiting the expression of the inflammatory pathways [81,82,83]. This indicates that the oil’s antimicrobial activity results from the synergistic action of more than one compound.

The results demonstrated in Table 8 clearly show that both nonanal and cis-rose oxide interact better with the four protein targets, showing differential binding energies and complex formation. Moreover, it is interesting to predict how these compounds could potentially contribute to the multiple functions of SN in serving as antimicrobial and antioxidant therapeutic agents. Tyrosyl–tRNA synthetase possesses a binding pocket delineated by the amino acid residues Ala37, Asp38, Thr40, Ala41, Ser43, Leu44, His48, and Ile101 [84]. Nonanal exploits the amino acids within this region to exert both polar (His50) and non-polar (alkyl-typed hydrophobic bonds) with this protein (Figure 1a). Both nonanal and cis-rose oxide exhibited hydrophilic solid and hydrophobic interactions with penicillin-binding protein 2 (Figure 1b,c) and 1,3-β-glucan synthase (Figure 1g,h), thus potentially exercising inhibition of bacterial and fungal cell wall synthesis, thereby halting wall formation and ultimately leading to the death of the bacteria and fungi [80]. Virulence microorganisms have been reported to use catalase and glutathione peroxidase to detoxify reactive oxygen species, which is increased by the phagocytotic pathway [83,84]. Interestingly, similar to the results of other studies, the docking study revealed mechanistic insight into how 2-Pentadecanone, 6,10,14-trimethyl-, trans-rose oxide, and nonanal inhibits these antioxidant enzymes (Figure 1d–g). The protein prenylation pathway is important for the post-translation lipid modification of key cellular proteins in fungi, and arresting this process is central to limiting their infection to humans [85]. Therefore, in this study, nonanal is shown to possess strong potential binding sites for protein geranylgeranyltransferase-I (Figure 1j) in comparison to other identified compounds in SNEO. Provisionally, understanding the inhibitory potential of the compounds is supported by their strong and equally binding interaction with key protein targets crystallized from microorganisms similar to those utilized in determining the antimicrobial and antioxidant potential of the SNEO in vitro.

5. Conclusions

The present study showed the bioactive characteristics of Sambucus nigra oil, revealing a balanced composition and major biological activities. Fatty acid analysis showed a profile mainly composed of oleic acid (32.8%), palmitic acid (26.6%), and linolenic acid (12.8%), which confirmed its stability and functional properties. GC–MS identified the presence of volatile compounds such as nonanal (4.36%), cis-rose oxide (3.87%), and trans-rose oxide (1.74%), which are known for their antimicrobial and antioxidant roles in SNEO. Antioxidant activity, by means of DPPH radical scavenging, showed significant inhibition at an IC₅₀ value equivalent to 2.52 mg/mL—comparable to that of ascorbic acid. The antimicrobial activity was assayed to show moderate inhibitory effects, with C. albicans being the most sensitive (IC₅₀ = 3.08 mg/mL), followed by E. coli (IC₅₀ = 2.90 mg/mL). While the Gram-negative bacteria generally revealed a higher extent of resistance, S. pyogenes and S. aureus showed moderate susceptibility at high concentrations. These results were complemented by molecular docking studies, which revealed strong interactions between key volatile compounds, such as nonanal and cis-rose oxide, and microbial proteins. Such interactions could inhibit cell wall synthesis and the enzymatic activity necessary for microbial viability.

This research finding opens the therapeutic window for the use of SNEO as a natural antimicrobial and antioxidant agent. Its efficacy against fungal strains, specifically C. albicans, along with its antioxidant features, suggests a range of uses in treating microbial infections and oxidative stress. Future research should be directed at the practical applications of this compound, including clinical trials and formulation development, and its importance in sustainable agriculture and alternative medicine.