Integrating network pharmacology to investigate the molecular mechanism underlying the antibacterial action of lavender essential oil

doi:10.21203/rs.3.rs-7564643/v1

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Research Article

Integrating network pharmacology to investigate the molecular mechanism underlying the antibacterial action of lavender essential oil

https://doi.org/10.21203/rs.3.rs-7564643/v1

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Background

Lavender (Lavandula), a perennial plant in the Lamiaceae family, produces essential oil with natural antibacterial activity. However, the precise antibacterial mechanism of this essential oil remains incompletely understood.

Methods and Materials

This study analyzed essential oils from Lavandula × intermedia (Xinxun 4) and three Lavandula angustifolia cultivars ( 'Xinxun 1', 'Xinxun 2', 'Xinxun 3') using GC/MS. An integrated approach combined network pharmacology, molecular docking simulations, and in vitro experiments was used to elucidate the antibacterial potential. Intersection analysis compared 1602 disease-related targets with 440 lavender essential oil targets. Protein-protein interaction (PPI) network analysis identified 27 overlapping targets, which underwent gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analyses to uncover biological functions and pathways. Molecular docking assessed binding affinity between key constituents of lavender essential oil (LEO) and target proteins of human being. In vitro experiments evaluated the inhibitory effects of LEO on Staphylococcus aureus.

Results

GC/MS identified 68 components in LEO. Network analysis revealed 136 potential antibacterial targets, with PPI analysis narrowing these to 27 core targets. GO/KEGG analyses indicated relevant biological functions and pathways. Molecular docking demonstrated high-affinity binding of the lavender constituents Neryl acetate and Geranyl acetate to the proteins PTGS2 and GSK3β, respectively, establishing them as core targets. In vitro experiments confirmed that LEO exhibited 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity and could induce malondialdehyde (MDA) accumulation in Staphylococcus aureus, as well as effectively inhibit bacterial growth.

Conclusion

These findings elucidate that lavender essential oil inhibits Staphylococcus aureus through the induction of oxidative stress, as evidenced by MDA accumulation, and via antioxidant activity, demonstrated by DPPH radical scavenging. Additionally, its active components may exert antibacterial effects by targeting potential human molecular targets, including PTGS2 and GSK3β.

Lavender

essential oil

antibacterial

network pharmacology

molecular docking

Pathogenic microorganisms pose a significant threat to both human and animal health. Typically, these microorganisms do not impact the environment and are considered components of the normal biological community [1]. However, when environmental conditions become highly conducive to their growth, they can proliferate rapidly, leading to environmental contamination. This proliferation may facilitate the spread of pathogens through air, water, and other media, resulting in infections in various organisms and the emergence of diseases, such as food borne illnesses, skin infections, pneumonia, and sepsis, which can even lead to death [2]. Common pathogenic bacteria include Escherichia coli(E.coil), Staphylococcus aureus (S. aureus), Salmonella spp., and Vibrio cholerae (V.cholera) [3]. Antibiotics are routinely employed as microbial control measurements, however, pathogenic microorganisms can easily mutate during transmission. Prolonged use of antibiotics can lead to the development of drug-resistant genes in these pathogens, creating serious biosafety concerns [4]. To address the issue of antibiotic resistance, various natural antibacterial compounds, such as essential oils, have been investigated for applications in the food industry and aquaculture [5, 6]. Essential oils, derived from aromatic plants, are complex mixtures of secondary metabolites that are widely recognized as natural antimicrobial agents. Due to their effective antibacterial and antioxidant properties, as well as their absence of adverse side effects, essential oils are considered viable alternatives to chemical additives in food and cosmetic products [7, 8].

Lavender stands as one of the world's premier aromatic plants [9, 10], distinguished by its rich and intense fragrance that has earned it the prestigious titles 'aromatic herb' and 'queen of herbs' [11]. The plant's characteristic aroma is primarily attributed to its volatile organic compounds (VOCs) [12]. These VOCs are fundamental in driving consumer preference for plant essential oil fragrances and find extensive applications across medicine, aromatherapy, healthcare, and cosmetic industries [13]. Lavender essential oil (LEO), a volatile aromatic oil characterized by its distinctive fragrant scent, is extracted through steam distillation of flowering spikes or leaves (either fresh or dried), following methods similar to other aromatic plants [14]. The composition of LEO typically comprises complex mixtures of mono- and sesquiterpenes and their derivatives, including esters, alcohols, ketones, and oxides, along with other volatile compounds [15, 16]. The principal components include linalyl acetate, linalool, 1,8-cineole, terpinen-4-ol, β-ocimene, and camphor, among others [17, 18]. The concentration of these constituents can vary significantly among oils derived from different cultivars, with their relative proportions largely determining the oil's market value, practical applications, and aromatic profile [19, 20].

Lavender and its essential oil are widely used in the medical field for stimulating the nervous system, sedation, stress relief, and treating insomnia [21–23]. Additionally, it has skin disinfection properties, promotes wound healing [24], reduces scarring, and possesses strong preservative and antibacterial effects [25–27]. Various lavenders have similar ethnobotanical properties and major chemical constituents, however, there are some differences in the reported antibacterial activity for different species.

There are different lavender species distributed in Yili district, Xinjiang Uygur Autonomous Region. Lavandula angustifolia Mill. (L. angustifolia, shorted as LA), is a frost-resistant species with beautiful flower colors and a variety of attractive cultivars [21]. Its essential oil is primarily composed of linalyl acetate and linalool (accounting for approximately 30–50% of its constituents) [28]. These components exhibit mild antimicrobial and anti-inflammatory properties, with particularly notable inhibitory effects against Gram-positive bacteria such as S. aureus [29]. Lavandula latifolia Vill. (L. latifolia, shorted as LL) contains abundant 1,8-cineole and camphor (accounting for approximately 10–20%), demonstrating higher volatility and stronger irritant properties [28–30]. The camphor and cineole components show significant antimicrobial activity against respiration-associated pathogens including Streptococcus pneumoniae and certain fungal species [31]. Lavandula × intermedia (Xinxun 4) displays an intermediate chemical composition between L. angustifolia and L. latifolia, and which antimicrobial efficacy combines broad-spectrum activity with moderate potency of them [32]. Given the economic importance and functions in healthcare and cosmetic formulation, and considering that the chemical composition of essential oil varies substantially, different lavender cultivars may result in distinct biological effects, furthermore, their essential oils have been documented to exhibit potent antibacterial efficacy. Despite the well-documented therapeutic benefits of LEO, bioinformatics-based assessment is required to decipher the molecular mechanisms underlying its antibacterial action.

Network pharmacology-based analysis represents a promising computer-aided drug discovery approach that facilitates the understanding of complex interaction networks and pathways between disease targets and therapeutic agents [33]. Our previous studies have analyzed the potent antibacterial activity of LEO. To further investigate the underlying mechanism, we employ an integrated approach combining computational pharmacology and molecular docking simulation to gain valuable insights into compound (LEO) - protein target interactions at the atomic level, predicting ligand-receptor conformations, and finally elucidating the potential molecular mechanisms of action [34]. This study utilized gas chromatography-mass spectrometry (GC-MS) for the comprehensive identification and quantitative analysis of the chemical constituents of LEO. An integrated approach combining computational pharmacology and molecular docking was employed to explore the pharmacological efficacy of LEO, validate its associated targets, and elucidate potential molecular mechanisms of action. Subsequently, in vitro experiments were conducted to further evaluate the inhibitory effects of LEO from different lavender cultivars against S. aureus. These findings may provide a valuable theoretical foundation for future research.

2.1 Materials

2.1.1. Chemicals

Ampicillin and Kanamycin were purchased from Yeasen (Shanghai), 2,2-diphenyl-1-picryl-hydrazyl (DPPH·) and L-ascorbic acid, dimethyl sulfoxide (DMSO) were purchased from Solarbio (Beijing). The essential oils of four varieties were prepared and by courtesy of Guantong Biology Group Co. Ltd. (Yili, Xinjiang, China). All other solvents used were of analytical grade.

2.1.2 Plants

Three Lavandula angustifolia cultivars, 'Xinxun 1, 'Xinxun 2', and Xinxun 3 ', along with Lavandula × intermedia (Xinxun 4), had been harvested in the Yili Tianshan Huahai tourism scenic area, Yining County, Xinjiang, China (81°98ʹ23ʺE, 43°70ʹ0ʺN; 750 m). The four lavender cultivars had been formally identified by the Fourth Division Agricultural Science Research Institute of the Xinjiang Production and Construction Corps and Researcher Li Min. Voucher specimens had been deposited in the herbarium of the same institute, and their official identities had been confirmed by the Crop Variety Registration Certificates (Nos. : Xindeng Lavender 2013-21 for Xinxun 1, 2013-22 for Xinxun 2, 2015-41 for 'Xinxun 3', and 2015-45 for Xinxun 4) issued by the Xinjiang Uygur Autonomous Region Non-Staple Crop Variety Registration Office. Sampling had been conducted on public land under the jurisdiction of Guantong Biology Group Co. Ltd., which had permitted the collection for research purposes.

2.2 Extraction, chemical characterization, and component identification of LEO

2.2.1. Preparation of LEO

LEO was extracted via steam distillation using fresh flower spikes collected at the early flowering stage. Post-harvest processing was performed to remove impurities and mechanical fragmentation of flower spikes to enhance the steam penetration. The steam was introduced at 80–100°C, and the resulting vapor mixture (containing volatile compounds and water vapor) was condensed and separated. The crude LEO was filtered and stored in amber glass bottles under light-restricted, sealed, and temperature-controlled conditions (15–20°C) to prevent degradation.

2.2.2. Gas chromatography/mass spectrometry (GC/MS)

A Varian 3800 gas chromatograph coupled with a Saturn 2200 ion trap mass spectrometer was used for chemical characterization. Separation was achieved on a 30 m × 0.25 mm capillary column with helium carrier gas (1 mL·min^-1). The temperature program included: 40°C for 3 min, then increased to 130°C (10°C per min), finally to 230°C (50°C per min), and held for 8 min. chromatographic retention indices and mass spectral profiles with authenticated reference compounds from an in-house repository, acquired under identical analytical conditions [35].The methyl nonanoate (used as the internal standard) and n-alkane standards (C7–C30) for the experiments were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

2.3 Network pharmacology-based prediction of antimicrobial targets and interaction network construction for LEO

2.3.1. Identification of potential targets of LEO

Based on our prior GC-MS analysis of four lavender LEO, sixty eight compounds were characterized, and which structural details were retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov/). Following initial screening via the SwissADME website, the SMILES notations of these compounds were utilized for target prediction using the SwissTargetPrediction website, and yielding 440 potential targets.

2.3.2. Drug targets for LEO

Targets associated with the inhibition of S. aureus were acquired from the GeneCards database (https://www.genecards.org/), the OMIM database (https://www.omim.org/), and the CTD database (https://ctdbase.org/). The intersection of all S. aureus inhibition-related targets was then obtained, and yielding 1,602 relevant targets for S. aureus inhibition.

2.3.3. Construction of protein-protein interaction and compound-target networks

Using Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html), an intersection analysis was performed between targets of LEO key component and S. aureus inhibition targets. These intersected genes represent potential targets for LEO in inhibiting S. aureus. Subsequently, protein-protein interaction (PPI) networks for the common targets were constructed using the STRING database (https://cn.string-db.org/). The resulting tsv files were downloaded and imported into Cytoscape software (versions 3.7.2 and 3.9.1) for visualization, yielding 27 hub targets through topological analysis. Through systematic association analysis, the 27 identified core targets were further correlated with the major bioactive components exhibiting significant concentration differences among the four LEO varieties to construct a comprehensive compound-target interaction network.

2.4 Multi-omics analysis of antimicrobial mechanisms of LEO

2.4.1. GO and KEGG analysis

Functional enrichment analysis of gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) pathways for the potential targets of LEO in inhibiting S. aureus was performed using the functional annotation tool within the DAVID database (https://david.ncifcrf.gov/). The resulting data were subsequently processed and visualized using the Bioinformatics platform (http://www.bioinformatics.com.cn/).

2.4.2. Construction of a disease-target-pathway network

A compound-target-pathway network was constructed in Cytoscape (version 3.9.1 and 3.7.2) by incorporating the bioactive constituents of LEO, their potential targets for inhibiting S. aureus, and relevant KEGG pathways. Within this network, nodes represent the bioactive constituents of LEO, genes/proteins, or pathways, while edges denote biological relationships between these molecular entities.

2.4.3. Molecular docking analysis

The PDB files for the co-crystallized ligands PTGS2 and GSK3B were retrieved from the PDB database (https://www.rcsb.org/). Additionally, SDF files for the two bioactive constituents of LEO Neryl acetate and Geranyl acetate were acquired from PubChem. Using OpenBabel (version 3.1.1), the SDF files were converted to mol2 format. The mol2 files of the ligands were then imported into AutoDockTools (version 1.5.7) for molecular docking. The resulting docking poses were visualized using PyMOL (version 3.0.3).

2.5 Antibacterial activity test

2.5.1 Test microorganisms and inoculation preparation

Following activation of S. aureus, a single colony was selected and inoculated into LB broth. The culture was incubated overnight at 37°C with shaking at 150 rpm. The bacterial suspension was subsequently adjusted to a concentration of 1×10⁶ CFU·mL^− 1 and stored for subsequent use.

2.5.2 Inhibition zone assay

The agar diffusion method was used to screen the antibacterial activity of LEO [36]. Bacterial suspensions (200 µL of each) were spread on LB solid medium, then the filter paper disks (about 6 mm in diameter) were placed to contact the surface of the medium, and then the extracted LEO (60 µL) was added onto the disk. The treated petri dishes were placed at 4°C for 1 h to allow the LEO to spread around the filter paper disk, then incubated at 37°C for 24 h under aerobic conditions, and then the diameter of the microbial growth-inhibition-zone around the sample disk was measured in millimetres with a vernier caliper. The Ampicillin (100 µg·mL^− 1) was used as the positive control.

2.5.3 Determination of the minimum inhibitory concentration (MIC)

The MIC of 'Xinxun 4' LEO against S. aureus had been determined using the agar plate method. LEO had been diluted in dimethyl sulfoxide (DMSO) to obtain a series of concentrations (100%, 50%, 25%, 12.5%, and 6.25%) via two-fold dilution. Sterilized LB medium (15 mL per tube) had been cooled to 55 ℃, supplemented with 100 µL of each LEO dilution, mixed thoroughly, and poured into plates. After solidification, the plates had been inoculated with 100 µL of incubated at 37°C for 16–18 h. Colonies had been enumerated post-incubation. The antibacterial rate had been calculated as follows: Antibacterial rate (%) = (C − T) / C × 100%, where C and T had represented the mean colony counts of the control and treatment groups, respectively. Bacterial growth had been recorded as positive (+) for growth and negative (–) for inhibition. The MIC had been defined as the lowest LEO concentration that had completely prevented visible growth. All tests had been performed in triplicate with three independent replicates, and the results had been expressed as mean ± standard error.

2.6 DPPH· scavenging ability and malondialdehyde (MDA) assays

The antioxidant potential of four LEO was quantitatively assessed using the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH·) radical as a spectroscopic probe [37, 38]. DPPH· assay was performed using a kit from Solarbio (Beijing Solarbio Science & Technology Co., Ltd.). The working solution and LEO supernatant had been mixed in a gradient manner in a 96-well plate according to the kit instructions, with blank control and positive control groups having been set up respectively. Following 30 min incubation under dark conditions at 25 ± 1°C, spectrophotometric absorbance at 515 nm was recorded using a microplate reader (Multiskan GO 1510, Thermo Fisher Scientific Dy Ratastie 2, FI-01620 Vantaa, Finland). The free radical scavenging activity had been calculated according to the method provided in the instruction manual. MDA content was determined using a kit from Sangon Biotech (Shanghai Sangon Biological Engineering Co. Ltd.). All procedures were carried out in accordance with the manufacturer's instructions.

2.7 Statistical analysis

All analyses were performed in triplicate. Data were processed using one-way analysis of variance (ANOVA) followed by Duncan's multiple range test using GraphPad Prism software (version 10.1, GraphPad Software, La Jolla, CA, USA). Significant differences (P ≤ 0.05) were determined using Tukey's test for MDA content, free radical scavenging activity, and antibacterial activity assays were determined using Tukey's test.

3.1 Analysis of the chemical composition of LEO from four varieties

Empirical studies demonstrate significant interspecific variations in aromatic profiles among Lavandula cultivars, with these chemotypic differences directly modulating antimicrobial efficacy through differential regulation of bioactive metabolite synthesis pathways. The chemical compositions of LEO collected from the 'Xinxun 1', 'Xinxun 2', 'Xinxun 3' and 'Xinxun 4' were analyzed using GC-MS. Approximately 68 components were identified, and the main representative compounds of these essential oils included linalool, linalyl acetate, camphor, etc. The compositions (relative percentage in total, %) of four LEOs were present in Table S1.

Chromatographic profiling revealed distinct phytochemical compositions among the evaluated LEO. 'Xinxun 1' exhibited a monoterpenoid-dominated profile, with linalool (31.58%) and linalyl acetate (28.07%) as predominant constituents, accompanied by minor quantities of camphor (0.23%) and lavandulol (2.41%). The major components of 'Xinxun 3' LEO were characterized by elevated linalyl acetate (30.04%) and lavandulyl acetate (3.65%), alongside linalool (38.08%) and trace sesquiterpenoids (e.g., borneol: 0.65%). In contrast, 'Xinxun 4' LEO displayed a sesquiterpenoid-biased profile, marked by significantly elevated camphor (12.00%) and borneol (3.83%), with comparatively reduced linalool (24.13%) and linalyl acetate (20.43%). 'Xinxun 2' chemotype reaffirmed species-specific patterns, quantifying linalool (32.19%) and linalyl acetate (35.97%) as principal components, with minimal camphor content (0.09%). According to the analytical results, the linalool was found relatively higher content in L. angustifolia cultivars, while the camphor content was higher in Lavandula × intermedia species. These compositional discrepancies may correlate with their biological functions, e.g., the antimicrobial efficacy and therapeutic properties.

Table 1
The chemical composition (%), active site and corresponding target of tested lavender essential oil
Category	Xinxun 1 (L. angustifolia)	Xinxun 2 (L. angustifolia)	Xinxun 3 (L. angustifolia)	Xinxun 4 (Lavandula × Intermedia)	GI absorption	Log K_p (skin permeation) (cm/s)	Target	Common name
Monoterpenes
(-)-alpha-Pinene	5.43	0.15	0.23	0.58	Low	-3.95	Estrogen receptor alpha	ESR1
Linalool, (+-)-	0.3	32.19	38.08	24.13	High	-5.13	Heme oxygenase 1 (by homology)	HMOX1
							Tyrosine-protein kinase JAK2	JAK2
							Cyclooxygenase-2	PTGS2
Isopulegol	0.18	3.22	4.74	2.18	High	-5.15	Heme oxygenase 1 (by homology)	HMOX1
Lavandulol	0.09	0.28	0.65	0.45	High	-5.10	Tyrosine-protein kinase JAK2	JAK2
							Heme oxygenase 1 (by homology)	HMOX1
							Protein kinase C alpha	PRKCA
Geraniol	0.01	0.19	0.05	0.05	High	-4.71	Tyrosine-protein kinase JAK2	JAK2
							Cyclooxygenase-2	PTGS2
							Protein kinase C alpha	PRKCA
							Heme oxygenase 1 (by homology)	HMOX1
Cyclohexanol, 5-methyl-2-(1-methylethenyl)-	0.2	4.79	5.27	0.66	High	-5.15	Heme oxygenase 1 (by homology)	HMOX1
Terpenoid Derivatives
Linalyl acetate	0.27	35.97	30.42	20.43	High	-4.71	Cathepsin (B and K)	CTSB
							Heme oxygenase 1 (by homology)	HMOX1
							Epidermal growth factor receptor erbB1	EGFR
							Tyrosine-protein kinase JAK2	JAK2
							Heat shock protein HSP 90-beta	HSP90AB1
Isopulegyl acetate	0.23	5.94	4.68	1.9	High	-4.99	Heme oxygenase 1 (by homology)	HMOX1
							Cyclooxygenase-2	PTGS2
							Epidermal growth factor receptor erbB1	EGFR
Lavandulyl acetate, (+/-)-	0.13	0.09	3.65	0.01	High	-4.94	Tyrosine-protein kinase JAK2	JAK2
							Cathepsin (B and K)	CTSB
							Leukocyte common antigen	PTPRC
							Heme oxygenase 1	HMOX1
							MAP kinase p38 alpha	MAPK14
							Epidermal growth factor receptor erbB1	EGFR
							Heat shock protein HSP 90-alpha	HSP90AA1
							Proto-oncogene c-JUN	JUN
1-Octen-3-yl acetate	0.05	1.99	0.22	0.22	High	-5.12	Epidermal growth factor receptor erbB1	EGFR
							Heat shock protein HSP 90-beta	HSP90AB1
							Tyrosine-protein kinase JAK2	JAK2
							Cyclooxygenase-2	PTGS2
							Intercellular adhesion molecule-1	ICAM1
							Vascular cell adhesion protein 1	VCAM1
							MAP kinase p38 alpha	MAPK14
Hexyl isovalerate	0.03	0.14	0.21	0.12	High	-4.82	Epidermal growth factor receptor erbB1	EGFR
							Serine/threonine-protein kinase AKT	AKT1
							Heme oxygenase 1 (by homology)	HMOX1
							Protein kinase C alpha	PRKCA
							Intercellular adhesion molecule-1	ICAM1
							Vascular cell adhesion protein 1	VCAM1
							Beta amyloid A4 protein	APP
							Alpha-synuclein	SNCA
							MAP kinase p38 alpha	MAPK14
							Cytochrome P450 3A4	CYP3A4
							c-Jun N-terminal kinase 1	MAPK8
Neryl acetate	0.02	0.31	0.17	0.2	High	-4.63	Epidermal growth factor receptor erbB1	EGFR
							Cyclooxygenase-2	PTGS2
							Cathepsin (B and K)	CTSB
							Tyrosine-protein kinase JAK2	JAK2
							Glycogen synthase kinase-3 beta	GSK3B
							Matrix metalloproteinase 9	MMP9
							MAP kinase p38 alpha	MAPK14
Geranyl acetate	0.01	0.22	0.14	0.17	High	-4.63	Tyrosine-protein kinase SRC	SRC
							Cyclooxygenase-2	PTGS2
							Cathepsin (B and K)	CTSB
							Glycogen synthase kinase-3 beta	GSK3B
							Leukocyte common antigen	PTPRC
							Alpha-synuclein	SNCA
Hexyl formate	7.92	0.01	0.02	0.02	High	-5.35	Leukocyte common antigen	PTPRC
							Epidermal growth factor receptor erbB1	EGFR

3.2 Identification of antimicrobial targets associated with the composition of LEO

To investigate the potential mechanism underlying the inhibition effect of LEO on S. aureus, we systematically screened all the chemical compounds of LEO, and identified 440 potential targets for LEO bioactive constituents and 1602 targets associated with S. aureus inhibition. The Venn diagram analysis revealed 136 common targets resulting from the intersection of 304 bioactive compound targets and 1466 disease targets (Fig. 1A), which may represent the probable targets for LEO in inhibiting S. aureus. These 136 targets were imported into the STRING database to generate a PPI network, and then processed and visualized using Cytoscape software (versions 3.7.2 and 3.9.1). The resulting network comprised 27 nodes and 281 edges, where larger and darker nodes indicate higher interaction affinity (Fig. 1B). These core targets and key bioactive compounds involved terpenoid derivatives such as Geranyl acetate, Neryl acetate, and lavandulyl acetate. Among these, six monoterpenes and eight terpenoid derivatives exhibited significant predictable bioactivity. Based on the pharmacological relevance among these components, we further refined the core targets to 22 and constructed a comprehensive compound-target interaction network (Fig. 1C). The network pharmacology analysis revealed that these bioactive compounds possess unique polypharmacological properties. Notably, Geranyl acetate and Neryl acetate selectively modulate key proteins including prostaglandin-endoperoxide synthase 2 (PTGS2) and glycogen synthase kinase 3β (GSK3B), and these terpenoid derivatives established a multidimensional regulatory network by synergistically targeting the PTGS2-mediated inflammatory pathway and GSK3B-associated glucose metabolism, demonstrating a probable multi-pathway cooperative mechanism of action.

3.3 Analysis of networks and pathways of LEO compounds involved in

To elucidate the interactions between target proteins and their associated components, we performed analyses of GO terms and KEGG pathway using the DAVID bioinformatics resource. P values were adjusted via the Benjamini-Hochberg correction method, with significant terms (P ≤ 0.05) selected across biological process (BP), molecular function (MF), and cellular component (CC) categories. The top 10 significantly enriched terms related to S. aureus inhibition were present in Fig. 2A. GO functional analysis revealed that the primary targets of LEO involved in the enhancement of immune cell cytotoxicity, barrier defense and coagulation-mediated anti-infection mechanisms, intracellular signaling coordination for microbial killing, direct pathogen clearance through key enzymatic activities. KEGG pathway analysis identified 19 of S. aureus inhibition-associated signaling pathways, with the top 10 visualized in Fig. 2B, which were categorized as: pathogen recognition (3 pathways), inflammatory signal amplification (2 pathways), T-cell polarization (2 pathways), effector clearance mechanisms (1 pathway), cross-pathway regulatory hubs (2 pathways).

Based on the above data, a comprehensive ‘compound-target-pathway’ interaction network was constructed (Fig. 2C) that integrated 22 core target proteins with 10 key signaling pathways. Analysis revealed that 17 significantly enriched gene targets specifically interact with two major classes of bioactive compounds, i.e., monoterpenes (6 compounds) and terpenoid derivatives (8 compounds), clearly delineating a multi-layered ‘component-target-pathway’ regulatory network. Further KEGG pathway enrichment analysis identified three immunologically relevant pathways critical for host defense, i.e., (1) the C-type lectin receptor signaling pathway, which mediates pathogen recognition and macrophage bactericidal activity through NADPH oxidase-dependent ROS burst mechanisms; (2) the T-cell receptor signaling pathway that drives Th17 cell differentiation and IL-17-dependent neutrophil chemotaxis via the NF-κB/AP-1 transcriptional regulatory network; (3) the TNF signaling pathway that enhances neutrophil transendothelial migration by upregulating endothelial adhesion molecules (ICAM-1, VCAM-1) and pro-inflammatory cytokines (TNF-α, IL-6) through cascade amplification. These pathways collectively involve 30 core regulatory genes. In addition, two key bioactive components with significant chemotypic variations, i.e., Neryl acetate and Geranyl acetate, were also identified (Table 1). Through integrated network topology analysis and molecular docking validation, we further established interaction profiles between these compounds and 11 pathway-critical targets (Fig. 2D), and identified two hub targets - PTGS2 and GSK3β, by applying stringent "multi-component multi-target" screening criteria (requiring candidate targets to bind both core components simultaneously). This interaction network suggests a probable molecular mechanism by which LEO core components - Neryl acetate and Geranyl acetate - coordinately target PTGS2 and GSK3β to modulate three key immune pathways, thereby inhibiting S. aureus infection.

3.4 Molecular docking analysis

Based on the above data, molecular docking analyses were performed targeting the core anti-inflammatory and metabolic regulatory proteins PTGS2 and GSK3β with two major terpenoid constituents of LEO-Neryl acetate and Geranyl acetate. The results imply high-affinity binding for all protein-ligand complexes, with calculated binding energies (ΔG) ranging from − 5.88 to -5.59 kcal·mol^− 1 (ΔG ≤ -5.59 kcal·mol^− 1), indicative of spontaneous binding (ΔG < 0) and thermodynamic stability (|ΔG| >5 kcal·mol^− 1 signifying strong binding) (Table 2). Target-specific binding modes suggest distinct inhibition mechanisms: For PTGS2 (PDB: 5F19), Geranyl acetate anchored via hydrogen bonds to Asn375 (2.4 Å) and Arg376 (2.9 Å and 2.2 Å), sterically blocking arachidonic acid access to the catalytic cleft (Fig. 3A), while Neryl acetate simultaneously engaged Tyr130 (2.3 Å), Lys137 (2.4 Å), and the dimerization interface residue Lys546 (1.9 Å and 2.2 Å), achieving dual-site inhibition (Fig. 3B). For GSK3β (PDB: 1H8F), Geranyl acetate formed a triple hydrogen-bond network within the ATP pocket with hinge residue Val214 (2.0 Å and 2.1 Å), catalytic lysine Lys205 (2.2 Å), and Arg180 (2.7 Å and 1.8 Å), enabling direct competitive inhibition of phosphate transfer (Fig. 3C). In contrast, Neryl acetate-constrained by steric conformation and exhibited weaker hydrogen bonding (average bond length increased by 0.15 Å), resulting in significantly reduced binding affinity (-5.59 kcal·mol^− 1) relative to Geranyl acetate (ΔG = 0.29 kcal·mol^− 1) (Fig. 3D).

Table 2
Basic information on the docking of Neryl acetate and Geranyl acetate to target protein molecules
Molecular name	Target	PDB ID	Residue involved in H bonding	H-bond length (Å)	Binding energy (kcal·mol^− 1)
Geranyl acetate	PTGS2	5F19	ASN-375; ARG-376	2.4; 2.9; 2.2	-5.63
Neryl acetate	PTGS2	5F19	TYR-130; LYS-137; LYS-546	2.3; 2.4; 1.9; 2.2	-5.71
Geranyl acetate	GSK3B	1H8F	VAL-214; LYS-205; ARG-180	2.0; 2.1; 2.2; 2.7; 1.8	-5.88
Neryl acetate	GSK3B	1H8F	VAL-214; ARG-180; LYS-205	2.0; 2.1; 1.8	-5.59

3.5 Antimicrobial activity of LEO from four lavender varieties

The antibacterial efficacy of LEO from four varieties against S. aureus was evaluated by determining the inhibition zone (IZ) and the minimum inhibitory concentration (MIC). The results (Fig. 4A) showed that all four LEO exhibited inhibitory effects on the growth of S. aureus, with 'Xinxun 4' and 'Xinxun 3' LEO presenting the largest IZ (19 ± 0.8 mm and 17 ± 0.9 mm, respectively), while 'Xinxun 1' and 'Xinxun 2' displaying smaller IZ (13 ± 0.7 mm and 14 ± 0.8 mm, respectively). An antimicrobial coating experiment of 'Xinxun 4' LEO was used to determine the MIC based on the bacterial colony growth on agar plates (Fig. 4B-G). No colony was observed when the LEO volume fraction was ≥ 0.15%, which means that the MIC of LEO against S. aureus was about 0.15%. Our results indicate the significant antimicrobial activity of four cultivars’ LEO against S. aureus in the present study.

3.6 The free radical scavenging and antioxidant activity of LEO

The antioxidant potential analysis was conducted by the DPPH· radical scavenging assay. In this study, L-ascorbic acid (LAA) was used as a reference in antioxidant properties. Among the four LEO, 'Xinxun 1' variety displayed the greatest antioxidant activity against DPPH· (40.0 ± 1.6%), followed by 'Xinxun 4' (36.2 ± 2.6%), 'Xinxun 3' (33.7 ± 2.0%) and 'Xinxun 2' (29.7 ± 1.2%) (Fig. 5A). These results suggest that LEO and its components may be effective natural antioxidants.

Malondialdehyde (MDA) usually serves as a biomarker for assessing the extent of oxidative lipid degradation and cellular damage [39]. In this study, intracellular MDA level was quantified using the thiobarbituric acid (TBA) colorimetric assay. As shown in Fig. 5B, MDA content in S. aureus increased in all LEO-treated groups after 2 h of exposure. At 6 h, both the 1/2 MIC and MIC treatment groups exhibited significant increases in MDA content, whereas the control group maintained unchangeable throughout the experiment. The results suggest that the inhibitory effect of LEO to S. aureus may be induced by damaging the cell membrane to generate excess MDA, ultimately leading to apoptosis.

LEO possesses inherent antibacterial and anti-inflammatory properties [40, 41], serving as a natural antimicrobial agent with potential as an antibiotic alternative [42]. This study aims to investigate the bacteriostatic characteristics of LEO from different varieties, focusing on elucidating the potential mechanism of action of its active constituents. Although studies have demonstrated the antibacterial effect of different LEO, a comprehensive investigation into the underlying molecular mechanisms remains necessary.

Our study identified the chemical composition of LEO extracted from four primary varieties cultivated in Yili, Xinjiang, China. Significant variations were observed in the content of most compounds across the four lavender cultivars[43]. Neryl acetate and Geranyl acetate, constituents predicted for the bacteriostatic activity, exhibited the highest concentrations in the 'Xinxun 1' cultivar. The differential contents of the active constituent among different lavender cultivars may result in substantial variations in their biological efficacy[44].

Among the 68 compounds detected by GC-MS analysis in this study, 56 compounds were found to interact with 136 human target genes. The active components in lavender essential oil exert antibacterial effects through potential immunomodulatory mechanisms. KEGG pathway analysis further confirmed that these targets are closely associated with key signaling pathways, including the C-type lectin receptor (CLR), Tumor Necrosis Factor (TNF) signaling pathway, and T-cell receptor (TCR) signaling pathway[45]. We propose that the bioactive constituents in lavender oil may indirectly inhibit bacterial survival by modulating host immune responses, rather than through direct bactericidal activity. Specifically, these components potentially interact with human target proteins such as PTGS2 and GSK3β, thereby influencing downstream signaling cascades[46]. This interaction may enhance the recognition of pathogens via CLRs on innate immune cells (e.g., macrophages and dendritic cells), promoting phagocytosis and inflammatory responses. Concurrent activation of the TNF signaling pathway likely amplifies the inflammatory milieu and coordinates anti-bacterial effector functions, such as the induction of reactive oxygen species and containment of infection. Furthermore, modulation of the TCR signaling pathway may facilitate the activation and differentiation of T cells, promoting adaptive immune responses including Th1/Th17 cell-mediated macrophage activation and neutrophil recruitment, which are critical for clearing intracellular and extracellular bacteria, respectively[47]. Thus, lavender essential oil may orchestrate a multi-faceted anti-bacterial defense by synergistically enhancing both innate and adaptive immune pathways, highlighting its potential as an immunomodulatory agent against bacterial infections.

Molecular docking results suggest that Neryl acetate and Geranyl acetate, identified as core bioactive constituents inhibiting S. aureus in this study, regulate key downstream signaling pathways by binding to the target proteins PTGS2 and GSK3β (Fig. 2B). PTGS2 usually serves as a central regulator in both the CLR signaling pathway and the TNF signaling pathway. The former acts as a critical mediator in antibacterial defense, and precise modulation of CLR signal represents a pivotal direction for future anti-infective strategies [48]. The latter activates the NF-κB and MAPK pathways, driving TNF-α expression through phosphorylation and triggering an inflammatory cascade [49]. GSK3β functions as a core modulator of the TCR signaling pathway to mediate T-cell immunity, which may indirectly suppresses S. aureus infection by driving T-cell differentiation (into Th1, Th17, and cytotoxic T lymphocytes [CTLs]), enhancing phagocytic function, lysing infected host cells, and establishing immunological memory [50].

In our study, LEO of 'Xinxun 1' cultivar exhibited the strongest DPPH· radical scavenging capacity (40.0 ± 1.6%), significantly higher than 'Xinxun 4' (36.2 ± 2.6%) and 'Xinxun 3' (33.7 ± 2.0%) (Fig. 5B). This aligns with Sharma and Bhat's validation of the DPPH· as a rapid and reliable indicator for evaluating hydrogen-donating antioxidants [51]. However, 'Xinxun 1' showed the smallest inhibition zone diameter against S. aureus (13 ± 0.7 mm), whereas 'Xinxun 4' and 'Xinxun 3' exhibited significantly larger zones (19 ± 0.8 mm and 17 ± 0.9 mm, respectively) (Fig. 4A), suggesting a decoupling between antioxidant and antibacterial activities. The potent radical scavenging ability of 'Xinxun 1' may be attributed to its high content of terpenoid alcohols, particularly linalool (31.58%). Peana et al. have confirmed linalool's prominent DPPH· scavenging activity [52]. Conversely, the minimal camphor content (0.23%) of 'Xinxun 1' cultivar likely limited its antibacterial efficacy, for camphor exerts bacteriostatic effects by disrupting bacterial membrane integrity [53]. 'Xinxun 4' LEO (containing 12.00% camphor) may exert antimicrobial effects via camphor-induced oxidative stress, a widely reported mechanism among monoterpenes [54]. In this study, when S. aureus was treated with 'Xinxun 4' LEO at its minimum inhibitory concentration (MIC, 0.15% v/v), intracellular malondialdehyde (MDA, a terminal product of lipid peroxidation) increased progressively over time, becoming significantly higher than controls at 6 h (P < 0.05) (Fig. 5A). Usually the elevated MDA is used as a classic biochemical marker of ROS-mediated bacterial death [55]. MIC determination (Fig. 4B–G) confirmed that 0.15% 'Xinxun 4' LEO completely inhibited S. aureus growth, while colonies re-emerged at concentrations ≤ 0.075%. This trend seemingly correlated with MDA accumulation kinetics (persistent MDA increase in high-concentration groups). Such concentration-dependent bactericidal activity aligns with recent findings that the antimicrobial potency of LEO directly relates to their capacity to disrupt intracellular redox homeostasis, as demonstrated in studies on the essential oil of Thymbra spicata against food borne pathogens including S. aureus [56]. Network pharmacology-based predictions further analyzed that neryl acetate and geranyl acetate in LEO can interact with human target proteins (e.g., PTGS2 and GSK3β), thereby influencing downstream signaling cascades—including the CLR, TNF, and TCR signaling pathways—ultimately modulating host immune responses to indirectly inhibit bacterial survival [57].

This study conducted a preliminary investigation into the antibacterial mechanism of lavender essential oil (LEO). We predicted PTGS2 and GSK3B as the target proteins in human which exhibit strong binding affinity to Neryl acetate and Geranyl acetate, respectively, of two components in LEO. By such bindings, they may regulate the C-type lectin receptor signaling pathway, TNF signaling pathway, and T cell receptor signaling pathway, thereby inhibiting bacterial growth. In vitro antibacterial activity assays confirmed that LEO extracted from all four lavender cultivars exhibited antibacterial activity. Notably, LEO from the 'Xinxun 4' and 'Xinxun 3' cultivars displayed the most potent inhibitory effects, for both of their LEO produced significantly larger inhibition zones compared to those from the 'Xinxun 1' and 'Xinxun 2' cultivars. Interestingly, 'Xinxun 1' LEO exhibited better antioxidant capacity, suggesting potential synergistic interactions among its components. The antioxidant and antibacterial inhibition properties of the LEO of four cultivars may contribute to diverse applications in cosmetic formulations, as well as in the therapeutic and food industries.

Ethics approval and consent to participate

The collection of the plant material used in the present study was performed in accordance with the relevant collection and research permits granted by the institution.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Clinical trial number

not applicable

Funding

This work was Supported by Science Foundation for The Project for Ph.D Introduction of YiLi Normal University (grant no. 2023RCYG14), Youth Doctoral Talent of the 'Tianchi Yingcai' Project under the Third Batch of the '2 + 5' Key Talent Program in Xinjiang (grant no. 2025QNBS003) and University-Level Project of YiLi Normal University (grant no. 2024YSZD007).

Author Contribution

J.J.H. formulated the experimental design plan. Y.C.Z. was responsible for executing the experimental procedures and conducting data analysis. Y.L.Z. and J.N. drafted the manuscript. X.M.Z. and H.Y.L. reviewed and edited the manuscript. All authors have read and approved the final version of the manuscript.

Acknowledgments

Sincere thanks go to the anonymous Editors and reviewers for their helpful comments and suggestions.

Data Availability

The data supporting the findings of this study are available within the article and its accompanying supplementary materials.

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Integrating network pharmacology to investigate the molecular mechanism underlying the antibacterial action of lavender essential oil

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Abstract

Background

Methods and Materials

Results

Conclusion

Figures

1. Introduction

2. Materials and methods

2.1 Materials

2.1.1. Chemicals

2.1.2 Plants

2.2 Extraction, chemical characterization, and component identification of LEO

2.2.1. Preparation of LEO

2.2.2. Gas chromatography/mass spectrometry (GC/MS)

2.3 Network pharmacology-based prediction of antimicrobial targets and interaction network construction for LEO

2.3.1. Identification of potential targets of LEO

2.3.2. Drug targets for LEO

2.3.3. Construction of protein-protein interaction and compound-target networks

2.4 Multi-omics analysis of antimicrobial mechanisms of LEO

2.4.1. GO and KEGG analysis

2.4.2. Construction of a disease-target-pathway network

2.4.3. Molecular docking analysis

2.5 Antibacterial activity test

2.5.1 Test microorganisms and inoculation preparation

2.5.2 Inhibition zone assay

2.5.3 Determination of the minimum inhibitory concentration (MIC)

2.6 DPPH· scavenging ability and malondialdehyde (MDA) assays

2.7 Statistical analysis

3. Results

3.1 Analysis of the chemical composition of LEO from four varieties

3.2 Identification of antimicrobial targets associated with the composition of LEO

3.3 Analysis of networks and pathways of LEO compounds involved in

3.4 Molecular docking analysis

3.5 Antimicrobial activity of LEO from four lavender varieties

3.6 The free radical scavenging and antioxidant activity of LEO

4. Discussion

5. Conclusion

Declarations

Competing interests

Clinical trial number

Funding

Author Contribution

Acknowledgments

Data Availability

References

Additional Declarations

Supplementary Files

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